Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

ABSTRACT

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. patentapplication Ser. No. 12/316,370, entitled “IN VIVO UNNATURAL AMINO ACIDEXPRESSION IN THE METHYLOTROPHIC YEAST PICHIA PASTORIS” by Travis Younget al., filed Dec. 10, 2008; International Patent Application Serial No.PCT/US2008/013568, entitled “IN VIVO UNNATURAL AMINO ACID EXPRESSION INTHE METHYLOTROPHIC YEAST PICHIA PASTORIS” by Travis Young et al., filedDec. 10, 2008; and U.S. Provisional Patent Application Ser. No.61/208,141, entitled “Production of Carrier-Peptide Conjugates UsingChemically Reactive Unnatural Amino Acids, by Travis Young et al., filedFeb. 20, 2009; the contents of which are incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The invention was made with United States Government support under Grant

No. DE-FG03-00ER46051 from the United States Department of Energy,Division of Materials Sciences. The United States Government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates to the field of protein chemistry. Describedherein are methods for producing carrier polypeptide-target polypeptideconjugates wherein a first unnatural amino acid that has beenincorporated into the carrier polypeptide variant is reacted with asecond unnatural amino acid that has been incorporated into the targetpolypeptide variant. Compositions produced by these methods are alsodescribed.

BACKGROUND OF THE INVENTION

A variety of limitations impede the development of peptides fortherapeutic use. For example, therapeutic peptides generally exhibit lowstability in vivo and are often rapidly cleared, e.g., within severalminutes to a few hours, via chemical or enzymatic degradation followingtheir administration to a subject, i.e., before any therapeutic effectcan be achieved. Consequently, low bioavailability can necessitatefrequent administration of the peptide, often by injection, at fairlyhigh doses to maintain activity. Such high doses can lead to undesiredside effects. Furthermore, the delivery of therapeutic peptides can berestricted by the selective permeability of membrane barriers (e.g.,intestinal and blood-brain barriers). To promote their delivery intocells, to increase their half-life, and/or to maintain their activities,polypeptides of interest, e.g., small therapeutic polypeptides, can becovalently coupled to carrier polypeptides, e.g., any of a variety ofpolypeptides that can have a high affinity for a specific ligand orgroup of ligands e.g., sugars, nucleosides, salts, amino acids, fattyacids, or other molecules. Carrier polypeptides typically facilitate thetransport of such ligands, e.g., into subcellular compartments, inextracellular fluids (e.g., in the blood) or across cell membranes.Beneficially, carrier proteins can thus promote the delivery ofcovalently linked target polypeptides into cells, reduce their toxicity,and/or prolong their stability and/or activity following theadministration of the carrier-target conjugate to a subject.

Current methods for chemically coupling small peptides to carrierpolypeptides range from the use of non-specific reagents, e.g.,glutaraldehyde or carbodiimide activated N-hydroxysuccinimide esters, tohighly specific heterobifunctional crosslinkers that can circumvent theformation of carrier polypeptide-carrier polypeptide ortarget-polypeptide-target polypeptide conjugates. However, such reagentscan only be used to modify a limited number of amino acid residues(e.g., amino acids comprising amine, keto, thiol, sulfhydryl, orcarboxyl groups). Using such crosslinkers to conjugate a carrierpolypeptide to a target polypeptide can perturb the conformation of thecarrier polypeptide, the target polypeptide, or the resultingcarrier-target polypeptide conjugate, thus decreasing the conjugate'sstability, biological activity, pharmacokinetic activity, etc. Couplingreactions that make use of these crosslinking reagents can produce aheterogeneous population of carrier-polypeptide-target polypeptideconjugates, decreasing manufacturing efficiency and complicating qualitycontrol.

What are needed in the art are methods and compositions for theefficient, cost-effective, large-scale production of homogenouspopulations of carrier polypeptide-target polypeptide conjugates. Theinvention provides these and other needs, as will be apparent uponreview of the following disclosure.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for making carrierpolypeptide-target polypeptide conjugates that can exhibit increasedbioavailability, pharmacological activity, biological activity,half-life, and/or immunogenicity. The current invention utilizes thedirect incorporation of first and second unnatural amino acids intocarrier polypeptides and target polypeptides, respectively. The firstand second unnatural amino acids present in the carrier and targetpolypeptide variants can then be reacted to produce the conjugates ofthe invention. Such conjugates can find therapeutic or pharmaceuticaluse, and they can be beneficially produced in a low-cost expressionsystem that is capable of producing biologically active heterologousproteins that comprise complex posttranslational modifications.

In one aspect, the invention provides methods of making a carrierpolypeptide-target polypeptide conjugate. The methods includeincorporating a first unnatural amino acid residue into a carrierpolypeptide during synthesis or translation of the carrier polypeptide,incorporating a second unnatural amino acid residue into a targetpolypeptide during synthesis or translation of the target polypeptide,and reacting the first and second residue to produce the carrierpolypeptide-target polypeptide conjugate. The first and second unnaturalamino acids can optionally be reacted via one or more of: anelectrophile-nucleophile reaction, a ketone reaction with a nucleophile,an oxime ligation, an aldehyde reaction with a nucleophile, a reactionbetween a carbonyl group and a nucleophile, a reaction between asulfonyl group and a nucleophile, an esterification reaction, a reactionbetween a hindered ester group and a nucleophile, a reaction between athioester group and a nucleophile, a reaction between a stable iminegroup and a nucleophile, a reaction between an epoxide group and anucleophile, a reaction between an aziridine group and a nucleophile, areaction between an electrophile and an aliphatic or aromatic amine, areaction between an electrophile and a hydrazide, a reaction between anelectrophile and a carbohydrazide, a reaction between an electrophileand a semicarbazide, a reaction between an electrophile and athiosemicarbazide, a reaction between an electrophile and acarbonylhydrazide, a reaction between an electrophile and athiocarbonylhydrazide, a reaction between an electrophile and asulfonylhydrazide, a reaction between an electrophile and a carbazide, areaction between an electrophile and a thiocarbazide, a reaction betweenan electrophile and a hydroxylamine, a reaction between a nucleophile ornucleophiles such as a hydroxyl or diol and a boronic acid or ester, atransition metal catalyzed reaction, a palladium catalyzed reaction, acopper catalyzed heteroatom alkylation reaction, a cycloadditionreaction, a 1,3, cycloaddition reaction, a 2,3 cycloaddition reaction,an alkyne-azide reaction, a Diels-Alder reaction, or a Suzuki couplingreaction. In preferred embodiments, the reactions in which conjugatesare produced optionally proceed with an efficiency of greater than 50%,greater than 70% or greater than 90%.

Incorporating the first unnatural amino acid into the carrierpolypeptide during translation can optionally comprise providing atranslation system that includes the first unnatural amino acid, anorthogonal tRNA-synthetase (O-RS), an orthogonal tRNA tRNA) that isspecifically aminoacylated by the O-RS with the first unnatural aminoacid, and a nucleic acid encoding the carrier peptide, wherein thenucleic acid comprises a selector codon that is recognized by theO-tRNA; and translating the nucleic acid, thereby incorporating thefirst unnatural amino acid into the carrier polypeptide duringtranslation. Optionally, the carrier polypeptide (or target polypeptide)can be produced during translation in a methylotrophic yeast cell, e.g.,Candida cell, a Hansenula cell, a Pichia cell, or a Torulopsis cell.

In particular embodiments of the methods, incorporating the firstunnatural amino acid into the carrier polypeptide results in an HSAvariant comprising the first unnatural amino acid e.g., an HSA variantcomprising a p-acetylphenylalanine. Optionally, the first unnaturalamino acid can be incorporated into the HSA variant during translation.For example, the first unnatural amino acid can optionally beincorporated into the HSA variant at amino acid position 37, whereinnumbering of amino acid position is relative to that of SEQ ID NO: 1.However, the carrier polypeptide into which the first unnatural aminoacid is incorporated can optionally be or be homologous to any of avariety of polypeptides including, but not limited to, e.g., an antibody(e.g., an OKT3 antibody, an HER2 antibody, etc.), an antibody fragment(e.g., an Fc, an Fab, an scFv, etc.), an albumin, a serum albumin, abovine serum albumin, an ovalbumin, a c-reactive protein, a conalbumin,a lactalbumin, a keyhole limpet hemocyanin (KLH), an ion carrierprotein, an acyl carrier protein, a signal transducing adaptor protein,an androgen-binding protein, a calcium-binding protein, acalmodulin-binding protein, a ceruloplasmin, a cholesterol estertransfer protein, an f-box protein, a fatty acid-binding proteins, afollistatin, a follistatin-related protein, a GTP-binding protein, aninsulin-like growth factor binding protein, an iron-binding protein, alatent TGF-beta binding protein, a light-harvesting protein complex, alymphocyte antigen, a membrane transport protein, a neurophysin, aperiplasmic binding protein, a phosphate-binding protein, aphosphatidylethanolamine binding protein, a phospholipid transferprotein, a retinol-binding protein, an RNA-binding protein, an s-phasekinase-associated protein, a sex hormone-binding globulin, athyroxine-binding protein, a transcobalamin, a transcortin, atransferrin-binding protein, and/or a vitamin d-binding protein.

In certain embodiments of the methods, incorporating a second unnaturalamino acid into the target polypeptide results in a TSP-1 variantcomprising the second unnatural amino acid, e.g., a TSP-1 variantcomprising a ε-(2-(aminooxy)acetyl)-L-lysine. Theε-(2-(aminooxy)acetyl)-L-lysine can optionally be incorporated into theTSP-1 variant at amino acid position 6 or at amino acid position 1,wherein numbering of amino acid position is relative to that of SEQ IDNO: 2. In other embodiments of the methods, incorporating a secondunnatural amino acid into the target polypeptide results in an ABT-510variant comprising the second unnatural amino acid, e.g., an ABT-510variant comprising a ε-(2-(aminooxy)acetyl)-L-lysine. Theε-(2-(aminooxy)acetyl)-L-lysine can optionally be incorporated into theABT-510 variant at amino acid position 6 or at amino acid position 1,wherein numbering of amino acid position is relative to that of SEQ IDNO: 3. Optionally, the ε-(2-(aminooxy)acetyl)-L-lysine is incorporatedinto the TSP-1 variant or the ABT-510 variant during synthesis. However,the target polypeptide(s) into which a second unnatural amino acid isincorporated can optionally be or be homologous to, e.g., a TSP-1, anABT-510, a glugacon-like peptide-1 (GLP-1), a parathyroid hormone (PTH),a ribosome inactivating protein (RIP), an angiostatin, an Exedin-4, anapoprotein, an atrial natriuretic factor, an atrial natriureticpolypeptide, an atrial peptide, a C-X-C chemokine, a T39765, a NAP-2, anENA-78, a gro-a, a gro-b, a gro-c, an IP-10, a GCP-2, a NAP-4, an a PF4,a MIG, a calcitonin, a c-kit ligand, a cytokine, a CC chemokine, amonocyte chemoattractant protein-1, a monocyte chemoattractantprotein-2, a monocyte chemoattractant protein-3, a monocyte inflammatoryprotein-1 alpha, a monocyte inflammatory protein-1 beta, a RANTES, anI309, an R83915, an R91733, a T58847, a D31065, a T64262, a CD40 ligand,a complement inhibitor, a cytokine, an epithelial neutrophil activatingpeptide-78, a GROΥ, a MGSA, a GROβ, a GROγ, a MIP1-α, a MIP1-β, anMCP-1, an epithelial neutrophil activating peptide, an erythropoietin(EPO), an exfoliating toxin, a fibroblast growth factor (FGF), an FGF21,a G-CSF, a gonadotropin, a growth factor, a Hirudin, an LFA-1, a humaninsulin, a human insulin-like growth factor (hIGF), an hIGF-I, anhIGF-II, a human interferon, an IFN-α, an IFN-β, an IFN-γ, aninterleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, anIL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a keratinocytegrowth factor (KGF), a leukemia inhibitory factor, a neurturin, a PDGF,a peptide hormone, a pleiotropin, a pyrogenic exotoxin A, a pyrogenicexotoxin B, a pyrogenic exotoxin C, a relaxin, a somatostatin, asuperoxide dismutase, a thymosin alpha 1, a human tumor necrosis factor(hTNF), a human tumor necrosis factor alpha, a human tumor necrosisfactor beta, a Ras, a Tat, an inflammatory molecule, a signaltransduction molecule, a bovine pancreatic trypsin inhibitor (BPTI),and/or a BP320 antigen, wherein the target polypeptide comprises thesecond unnatural amino acid. It will be appreciated that the precedinglist is not intended to be limiting on the embodiments herein.

For example, using the methods described above, a p-acetylphenylalaninecan be incorporated into an HSA variant at amino acid position 37 duringtranslation, wherein the numbering of amino acid position in the HSA isrelative to SEQ ID NO: 1, a ε-(2-(aminooxy)acetyl)-L-lysine can beincorporated into a TSP-1 variant at amino acid position 6 duringsynthesis, wherein the numbering of the amino acid position in the TSP-1is relative to SEQ ID NO: 2, and the p-acetylphenylalanine and theε-(2-(aminooxy)acetyl)-L-lysine can be reacted via oxime ligation toproduce an HSA-TSP-1 conjugate. Optionally, using the methods describedabove, a p-acetylphenylalanine can be incorporated into an HSA variantat amino acid position 37 during translation, wherein the numbering ofamino acid position in the HSA is relative to SEQ ID NO: 1, aε-(2-(aminooxy)acetyl)-L-lysine can be incorporated into an ABT-510variant at amino acid position 6 during synthesis, wherein the numberingof the amino acid position in the ABT-510 is relative to SEQ ID NO: 3,and the p-acetylphenylalanine and the ε-(2-(aminooxy)acetyl)-L-lysinecan be reacted via oxime ligation to produce an HSA-ABT-510 conjugate

In a related aspect, the invention provides carrier polypeptide-targetpolypeptide conjugates produced by the methods described above.Optionally, a carrier polypeptide-target polypeptide conjugate of theinvention displays a longer serum half-life than the target polypeptide.A conjugate of the invention can optionally include any one or more ofthe carrier polypeptides described above and any one or more of thetarget polypeptides described above. However, it will be appreciatedthat the conjugates of the invention are not limited to those comprisingsaid carrier and target polypeptides.

The conjugate can optionally include a carrier polypeptide that is anHSA variant comprising at least a first unnatural amino acid and atarget polypeptide that is a TSP-1 variant or an ABT-510 variantcomprising the second unnatural amino acid. The first unnatural aminoacid can optionally be a p-acetylphenylalanine that has beenincorporated into, e.g., amino acid position 37 of an HSA variant, e.g.,during translation, wherein the numbering of amino acid position isrelative to that of SEQ ID NO: 1. The second unnatural amino acid canoptionally be a ε-(2-(aminooxy)acetyl)-L-lysine that has beenincorporated into, e.g., amino acid position 6 of a TSP-1 variant, e.g.,during synthesis, wherein the numbering of amino acid position isrelative to that of SEQ ID NO:2. The second unnatural amino acid canoptionally be a ε-(2-(aminooxy)acetyl)-L-lysine that has beenincorporated into, e.g., amino acid position 6 of an ABT-510 variant,e.g., during synthesis, wherein the numbering of amino acid position isrelative to that of SEQ ID NO: 3.

For example, a conjugate of the invention can comprise a carrierpolypeptide that is an HSA variant comprising a p-acetylphenylalanine atamino acid position 37, wherein the numbering of amino acid position isrelative to that of SEQ ID NO: 1, and a target polypeptide that is aTSP-1 variant comprising a ε-(2-(aminooxy)acetyl)-L-lysine at amino acidposition 6, wherein the numbering of amino acid position is relative tothat of SEQ ID NO: 2. Alternatively, a conjugate of the invention cancomprise a carrier polypeptide that is an HSA variant comprising ap-acetylphenylalanine at amino acid position 37, wherein the numberingof amino acid position is relative to that of SEQ ID NO: 1, and a targetpolypeptide that is an ABT-510 variant comprising aε-(2-(aminooxy)acetyl)-L-lysine at amino acid position 6, wherein thenumbering of amino acid position is relative to that of SEQ ID NO: 3. Inthese embodiments, the conjugate is produced by reacting thep-acetylphenylalanine and the ε-(2-(aminooxy)acetyl)-L-lysine via oximeligation

Relatedly, the invention provides carrier polypeptide-target polypeptideconjugates that comprise a carrier polypeptide domain comprising a firstunnatural amino acid residue, and a target polypeptide domain comprisinga second amino acid residue, wherein the carrier polypeptide domain andthe target polypeptide domain are conjugated together through the firstand second unnatural amino acid residues. A carrier polypeptide domainof the conjugate can optionally comprise any of the carrier polypeptidesdescribed herein, and a target polypeptide of the conjugate canoptionally comprise any one (or more) of the target polypeptide variantsdescribed herein.

In particular embodiments, the carrier polypeptide domain of a carrierpolypeptide-target polypeptide conjugate comprises an HSA, the firstunnatural amino acid residue is a p-acetylphenylalanine, the targetpolypeptide domain comprises a TSP-1, and the second unnatural aminoacid residue is a ε-(2-(aminooxy)acetyl)-L-lysine. In other embodiments,the carrier polypeptide domain of a carrier polypeptide-targetpolypeptide conjugate comprises an HSA, the first unnatural amino acidresidue is a p-acetylphenylalanine, the target polypeptide domaincomprises an ABT-510, and the second unnatural amino acid residue is aε-(2-(aminooxy)acetyl)-L-lysine. Optionally, the first unnatural aminoacid residue is incorporated into a carrier polypeptide domain duringtranslation in a methylotrophic yeast cell, e.g., a Candida cell, aHansenula cell, a Pichia cell, or a Torulopsis cell. A carrierpolypeptide and target polypeptide can optionally be covalently coupledby reacting the first and second unnatural amino acids in any one ormore of the reactions described herein.

Cells comprising the carrier polypeptide-target polypeptide conjugatesdescribed herein are also provided by the invention. For example, such acell can optionally comprise a polypeptide-target polypeptide conjugatewherein the carrier polypeptide domain of the conjugate comprises an HSAvariant that comprises a first unnatural amino acid, e.g., ap-acetylphenylalanine. A cell of the invention can comprise apolypeptide-target polypeptide conjugate wherein the target polypeptidedomain of the conjugate comprises a TSP-1 variant that comprises asecond unnatural amino acid, e.g., a ε-(2-(aminooxy)acetyl)-L-lysine. Inother embodiments, a cell of the invention can comprise apolypeptide-target polypeptide conjugate wherein the target polypeptidedomain of the conjugate comprises an ABT-510 variant that comprises asecond unnatural amino acid, e.g., a ε-(2-(aminooxy)acetyl)-L-lysine

Kits are also a feature of the invention. For example, kits canoptionally contain any one or more compositions provided by theinvention. Alternatively or additionally, kits can contain reagents forthe synthesis of carrier polypeptides that comprise first chemicallyreactive unnatural amino acids and/or target polypeptides, e.g., smalltherapeutic peptides, that comprise second chemically reactive unnaturalamino acids. Such reagents can include, e.g., the reactive unnaturalamino acids, host cells, e.g., methylotrophic yeast cells that includeorthogonal translation system components suitable for the production ofcarrier polypeptides and/or target polypeptides comprising unnaturalamino acids, solutions in which to perform ligation reactions thatproduce the conjugates of the invention, reagents with which to producetherapeutic formulations comprising one or more conjugates of theinvention, media, etc. Kits of the invention can include additionalcomponents such as instructions to, e.g., construct a methylotrophicyeast strain that can express a carrier polypeptide and/or targetpolypeptide that comprises unnatural amino acids, perform a chemicalligation reaction to produce a carrier polypeptide-target polypeptideconjugate, etc. The kit can include a container to hold the kitcomponents, instructional materials for practicing any method or anycombination of methods herein, instructions for using cells (e.g.,methylotrophic yeast cells) provided with the kit, e.g., to produce acarrier and/or target polypeptide of interest that comprises achemically reactive unnatural amino acid at a selected amino acidposition.

Those of skill in the art will appreciate that the methods, kits andcompositions provided by the invention can be used alone or incombination. For example, the methods of the invention can be used toproduce the carrier polypeptide-target polypeptide conjugates of theinvention. One of skill will appreciate further combinations of thefeatures of the invention noted herein.

DEFINITIONS

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “an aminoacyl tRNA synthetase (RS)” optionallyincludes combinations of two or more RS molecules; reference to “carrierpolypeptide” or “a methylotrohpic yeast cell” optionally includes, as apractical matter, a plurality of that carrier polypeptide or manymethylotrophic yeast cells.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of or testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

Carrier polypeptide: The term “carrier polypeptide” refers to any one ofa variety of polypeptides that can be conjugated to a targetpolypeptide, e.g., using the methods provided herein. A carrierpolypeptide can be beneficially used, e.g., to promote the delivery ofcovalently linked target polypeptides into cells, to reduce theirtoxicity, and/or to prolong their stability and/or activity followingthe administration of the carrier-target conjugate to a subject.Preferred characteristics of carrier proteins can include highsolubility and long half-lives; however, it is not intended that acarrier polypeptide be limited by possessing or not possessing anyparticular biological activity. Commonly used carrier proteins includehuman serum albumin (HSA; 66 kDa), bovine serum albumin (BSA; 67 kDa),antibody fragments such as Fc (45 kDa), and keyhole limpet hemocyanin(KLH; 4.5×10⁵-1.3×10⁷ Da). In one useful example described below,covalently linking the therapeutic peptide ABT-510 to the carrierpolypeptide HSA can increase the peptide's serum half-life.

Carrier polypeptides can optionally be modified versions of suchpolypeptides, e.g., modified by inclusion of an unnatural amino acid. Acarrier polypeptide into which an unnatural amino acid has beenincorporated is referred to herein as a “carrier polypeptide variant.”

Cognate: The term “cognate” refers to components that function together,or have some aspect of specificity for each other, e.g., an orthogonaltRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS), inwhich the O-RS specifically aminoacylates the O-tRNA with an unnaturalamino acid.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using a specified molecule ororganism, or sequence information from the specified molecule ororganism. For example, a polypeptide that is derived from a secondpolypeptide can include an amino acid sequence that is identical orsubstantially similar to the amino acid sequence of the secondpolypeptide. In the case of polypeptides, the derived species can beobtained by, for example, naturally occurring mutagenesis, artificialdirected mutagenesis or artificial random mutagenesis. The mutagenesisused to derive polypeptides can be intentionally directed orintentionally random, or a mixture of each. The mutagenesis of apolypeptide to create a different polypeptide derived from the first canbe a random event, e.g., caused by polymerase infidelity, and theidentification of the derived polypeptide can be made by appropriatescreening methods, e.g., as discussed in references cited herein.Mutagenesis of a polypeptide typically entails manipulation of thepolynucleotide that encodes the polypeptide.

Encode: As used herein, the term “encode” refers to any process wherebythe information in a polymeric macromolecule or sequence string is usedto direct the production of a second molecule or sequence string that isdifferent from the first molecule or sequence string. As used herein,the term is used broadly, and can have a variety of applications. Insome aspects, the term “encode” describes the process ofsemi-conservative DNA replication, where one strand of a double-strandedDNA molecule is used as a template to encode a newly synthesizedcomplementary sister strand by a DNA-dependent DNA polymerase. Inanother aspect, the term “encode” refers to any process whereby theinformation in one molecule is used to direct the production of a secondmolecule that has a different chemical nature from the first molecule.For example, a DNA molecule can encode an RNA molecule, e.g., by theprocess of transcription incorporating a DNA-dependent RNA polymeraseenzyme. Also, an RNA molecule can encode a polypeptide, as in theprocess of translation. When used to describe the process oftranslation, the term “encode” also extends to the triplet codon thatencodes an amino acid. In some aspects, an RNA molecule can encode a DNAmolecule, e.g., by the process of reverse transcription incorporating anRNA-dependent DNA polymerase. In another aspect, a DNA molecule canencode a polypeptide, where it is understood that “encode” as used inthat case incorporates both the processes of transcription andtranslation.

To incorporate an unnatural amino acid: As used herein, “to incorporatean unnatural amino acid”, e.g., into a carrier or target polypeptide,refers to the direct addition of an unnatural amino acid to a growingpolypeptide chain during primary construction of the carrier or targetpolypeptide, e.g., via translation or chemical synthesis.

In response to: As used herein, the term “in response to” refers to theprocess in which an O-tRNA of the invention recognizes a selector codonand mediates the incorporation of an unnatural amino acid, which iscoupled to the tRNA, into a growing polypeptide chain.

Non-eukaryote: As used herein, the term “non-eukaryote” refers toorganisms belonging to the Kingdom Monera (also termed Prokarya).Non-eukaryotic organisms, e.g., prokaryotic organisms, are generallydistinguishable from eukaryotes by their unicellular organization,asexual reproduction by budding or fission, the lack of a membrane-boundnucleus or other membrane-bound organelles, a circular chromosome, thepresence of operons, the absence of introns, message capping and poly-AmRNA, and other biochemical characteristics, such as a distinguishingribosomal structure. The Prokarya include subkingdoms Eubacteria andArchaea (sometimes termed “Archaebacteria”). Cyanobacteria (the bluegreen algae) and mycoplasma are sometimes given separate classificationsunder the Kingdom Monera. Nevertheless, Eubacteria, Archaea,Cyanobacteria, and mycoplasma are all understood to be non-eukaryotes.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule,e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl-tRNAsynthetase (O-RS)) that functions with endogenous components of a cellwith reduced efficiency as compared to a corresponding molecule that isendogenous to the cell or translation system, or that fails to functionwith endogenous components of the cell. In the context of tRNAs andaminoacyl-tRNA synthetases, orthogonal refers to an inability or reducedefficiency, e.g., less than 20% efficiency, less than 10% efficiency,less than 5% efficiency, or less than 1% efficiency, of an orthogonaltRNA to function with an endogenous tRNA synthetase compared to anendogenous tRNA to function with the endogenous tRNA synthetase, or ofan orthogonal aminoacyl-tRNA synthetase to function with an endogenoustRNA compared to an endogenous tRNA synthetase to function with theendogenous tRNA. The orthogonal molecule lacks a functionally normalendogenous complementary molecule in the cell. For example, anorthogonal tRNA in a cell is aminoacylated by any endogenous RS of thecell with reduced or even zero efficiency, when compared toaminoacylation of an endogenous tRNA by the endogenous RS. In anotherexample, an orthogonal RS aminoacylates any endogenous tRNA a cell ofinterest with reduced or even zero efficiency, as compared toaminoacylation of the endogenous tRNA by an endogenous RS. A secondorthogonal molecule can be introduced into the cell that functions withthe first orthogonal molecule. For example, an orthogonal tRNA/RS pairincludes introduced complementary components that function together inthe cell with an efficiency, e.g., 45% efficiency, 50% efficiency, 60%efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90%efficiency, 95% efficiency, or 99% or more efficiency, as compared tothat of a control, e.g., a corresponding tRNA/RS endogenous pair, or anactive orthogonal pair.

Orthogonal aminoacyl tRNA synthetase: As used herein, an orthogonalaminoacyl tRNA synthetase (O-RS) is an enzyme that preferentiallyaminoacylates an O-tRNA with an amino acid in a translation system ofinterest. The amino acid that the O-RS loads onto the O-tRNA can be anyamino acid, whether natural, unnatural or artificial, and is not limitedherein. The synthetase is optionally the same as or homologous to anaturally occurring tyrosyl amino acid synthetase, or the same as orhomologous to a synthetase designated as an O-RS.

Orthogonal tRNA: As used herein, an orthogonal tRNA (O-tRNA) is a tRNAthat is orthogonal to a translation system of interest, where the tRNAis, e.g., (1) identical or substantially similar to a naturallyoccurring tRNA, (2) derived from a naturally occurring tRNA by naturalor artificial mutagenesis, (3) derived by any process that takes asequence of a wild-type or mutant tRNA sequence of (1) or (2) intoaccount, (4) homologous to a wild-type or mutant tRNA; (5) homologous toany example tRNA that is designated as a substrate for an orthogonaltRNA synthetase or (6) a conservative variant of any example tRNA thatis designated as a substrate for an orthogonal tRNA synthetase. TheO-tRNA can exist charged with an amino acid, or in an uncharged state.It is also to be understood that a “O-tRNA” optionally is charged(aminoacylated) by a cognate synthetase with an unnatural amino acid.Indeed, it will be appreciated that an O-tRNA of the invention isadvantageously used to insert essentially any unnatural amino acid intoa growing polypeptide, during translation, in response to a selectorcodon.

Polypeptide: A polypeptide is any oligomer of amino acid residues(natural or unnatural, or a combination thereof), of any length,typically but not exclusively joined by covalent peptide bonds. Apolypeptide can be from any source, e.g., a naturally occurringpolypeptide, a polypeptide produced by recombinant molecular genetictechniques, a polypeptide from a cell or translation system, or apolypeptide produced by cell-free synthetic means. A polypeptide ischaracterized by its amino acid sequence, e.g., the primary structure ofits component amino acid residues. As used herein, the amino acidsequence of a polypeptide is not limited to full-length sequences, butcan be partial or complete sequences. Furthermore, it is not intendedthat a polypeptide be limited by possessing or not possessing anyparticular biological activity. As used herein, the term “protein” issynonymous with polypeptide. The term “peptide” refers to a smallpolypeptide, for example but not limited to, from 2-25 amino acids inlength.

Preferentially aminoacylates: As used herein in reference to orthogonaltranslation systems, an O-RS “preferentially aminoacylates” a cognateO-tRNA when the O-RS charges the O-tRNA with an amino acid moreefficiently than it charges any endogenous tRNA in an expression system.That is, when the O-tRNA and any given endogenous tRNA are present in atranslation system in approximately equal molar ratios, the O-RS willcharge the O-tRNA more frequently than it will charge the endogenoustRNA. Preferably, the relative ratio of O-tRNA charged by the O-RS toendogenous tRNA charged by the O-RS is high, preferably resulting in theO-RS charging the O-tRNA exclusively, or nearly exclusively, when theO-tRNA and endogenous tRNA are present in equal molar concentrations inthe translation system. The relative ratio between O-tRNA and endogenoustRNA that is charged by the O-RS, when the O-tRNA and O-RS are presentat equal molar concentrations, is greater than 1:1, preferably at leastabout 2:1, more preferably 5:1, still more preferably 10:1, yet morepreferably 20:1, still more preferably 50:1, yet more preferably 75:1,still more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1or higher.

The O-RS “preferentially aminoacylates an O-tRNA with an unnatural aminoacid” when (a) the O-RS preferentially aminoacylates the O-tRNA comparedto an endogenous tRNA, and (b) where that aminoacylation is specific forthe unnatural amino acid, as compared to aminoacylation of the O-tRNA bythe O-RS with any natural amino acid. That is, when the unnatural andnatural amino acids are present in equal molar amounts in a translationsystem comprising the O-RS and O-tRNA, the O-RS will load the O-tRNAwith the unnatural amino acid more frequently than with the naturalamino acid. Preferably, the relative ratio of O-tRNA charged with theunnatural amino acid to O-tRNA charged with the natural amino acid ishigh. More preferably, O-RS charges the O-tRNA exclusively, or nearlyexclusively, with the unnatural amino acid. The relative ratio betweencharging of the O-tRNA with the unnatural amino acid and charging of theO-tRNA with the natural amino acid, when both the natural and unnaturalamino acids are present in the translation system in equal molarconcentrations, is greater than 1:1, preferably at least about 2:1, morepreferably 5:1, still more preferably 10:1, yet more preferably 20:1,still more preferably 50:1, yet more preferably 75:1, still morepreferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.

Selector codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; rare codons; codons derived fromnatural or unnatural base pairs and/or the like.

Suppression activity: As used herein, the term “suppression activity”refers, in general, to the ability of a tRNA, e.g., a suppressor tRNA,to allow translational read-through of a codon, e.g., a selector codonthat is an amber codon or a 4-or-more base codon, that would otherwiseresult in the termination of translation or mistranslation, e.g.,frame-shifting. Suppression activity of a suppressor tRNA can beexpressed as a percentage of translational read-through activityobserved compared to a second suppressor tRNA, or as compared to acontrol system, e.g., a control system lacking an O-RS.

Suppression efficiency can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused, e.g., a derivatized lacZ plasmid (where the construct has aselector codon in the lacZ nucleic acid sequence) is introduced intocells from an appropriate organism (e.g., an organism where theorthogonal components can be used) along with plasmid comprising anO-tRNA of the invention. A cognate synthetase can also be introduced(either as a polypeptide or a polynucleotide that encodes the cognatesynthetase when expressed). The cells are grown in media to a desireddensity, e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression can be calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatized lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, typically byallowing the incorporation of an amino acid in response to a stop codon(i.e., “read-through”) during the translation of a polypeptide. In someaspects, a selector codon of the invention is a suppressor codon, e.g.,a stop codon, e.g., an amber, ocher or opal codon, a four base codon, arare codon, etc.

Target polypeptide: The term “target polypeptide” refers to anypolypeptide of interest whose, e.g., bioavailability, pharmacologicalactivity, biological activity, half-life, immunogenicity, etc. ismaintained or improved when it is covalently linked to a carrierpolypeptide. A target polypeptide can optionally be a small therapeuticpeptide, e.g., TSP-1 or ABT-510. Alternatively or additionally, a targetpolypeptide can be immunogenic, derived from a foreign organism, aself-protein, or a synthetic polypeptide. It is not intended that atarget polypeptide be limited by possessing or not possessing anyparticular biological activity. Examples of small therapeutic peptidesthat can be used with the invention include, e.g., a TSP-1 polypeptideor a derivative thereof, an ABT-510, a glugacon-like peptide-1 (GLP-1),a parathyroid hormone (PTH), Exedin-4, and many others, e.g., as notedherein.

Target polypeptides can optionally be modified versions of suchpolypeptides, e.g., modified by inclusion of an unnatural amino acid. Atarget polypeptide into which an unnatural amino acid has beenincorporated is referred to herein as a “target polypeptide variant.”

Translation system: The term “translation system” refers to thecomponents that incorporate an amino acid into a growing polypeptidechain (protein). Components of a translation system can include, e.g.,ribosomes, tRNAs, synthetases, mRNA, O-RSs, O-tRNAs, and the like.

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue, that is not one of the 20 common naturally occurring aminoacids or the rare naturally occurring amino acids e.g., selenocysteineor pyrrolysine.

Variant: As used herein, the term “variant” refers to a polypeptide thatcomprises at least one chemically reactive unnatural amino acid and istypically derived from a corresponding “natural” polypeptide thatcontains no unnatural amino acids. For example, a carrier polypeptidevariant (or target polypeptide variant) is a mutant of a “natural”carrier polypeptide (or “natural” target polypeptide), which mutantcomprises a chemically reactive unnatural amino acid. The chemicallyreactive unnatural amino acid in a carrier polypeptide variant and/or atarget polypeptide variant can optionally replace a natural amino acidin the carrier and/or target polypeptide's primary sequence.Alternatively or additionally, one (or more) unnatural amino acid can beadded to the primary sequence of a carrier or target polypeptide. Thechemically reactive unnatural amino acid that is incorporated into acarrier or target polypeptide variant can be a conservative ornon-conservative replacement (as compared to the corresponding naturalamino acid in the “natural” carrier or target polypeptide that comprisesno unnatural amino acids).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides schematic illustrations of various plasmids that finduse with the invention.

FIG. 2 shows the results of experiments that were performed to determinethe fidelity and specificity with which the unnatural amino acidp-acetylphenylalanine is incorporated into HSA in response to a selectorcodon at amino acid position 37.

FIG. 3 shows the results of experiments that were performed to comparepromoters to optimize pApaRS expression and amber suppression.

FIG. 4 shows the results of experiments that were performed to determineamber suppression levels with P_(AOX2), P_(YPT1), P_(ICL1), P_(FLD1),P_(GAP), or P_(AOX1) driven aaRS, as assayed by rHSA_(E37pApa) levels inthe media.

FIG. 5 a shows a schematic of oxime ligation of ABT-510 peptide torHSA_(E37pApa). FIG. 5 b shows the results of MALDI mass spectrometrythat was performed to determine the extent of conjugation.

FIG. 6 depicts experiments performed to illustrate that the orthogonaltranslation system of the invention can be used to incorporate unnaturalamino acids other than pApa (e.g., Structures 3-9) into rHSA_(E37X) inP. pastoris.

FIG. 7 depicts the results of PCR performed on 4 transformants todetermine whether pPIC3.5 k and pREAV cassettes were successfullyincorporated into GS200-rHSA_(E37X)/pREAV-P_(ADH1)-PApaRS.

FIG. 8 depicts western blots for pApaRS-_(His6x) in four separate clonesof GS200-rHSA_(E37X)/pREAV-P_(ADH1)-pApaRS from a single transformation.No pApaRS protein was detectable.

FIG. 9 depicts the results of an experiment that was performed todetermine whether rHSA_(E37X) is expressed more robustly in a P.pastoris Mut⁺ mutant.

FIG. 10 depicts the results of PCR that was performed to amplify variousP. pastoris gene promoters.

FIG. 11 provides a bar graph representation of the results of FIG. 4showing amber suppression in rHSA_(E37pApa) as a function of thepromoter driving pApaRS production.

FIG. 12 shows a protein gel on which 25 μl of BSA standards orunpurified rHSA_(WT) media from test protein expressions were run.

FIG. 13 shows LC-MS/MS of chymotrypsin-digested rHSA_(E37DMNB-C) proteinfrom lane 2 of FIG. 6 f.

FIG. 14 provides the sequences of oligonucleotide primers that are usedfor strain and plasmid construction in Example 1.

DETAILED DESCRIPTION OF THE INVENTION Overview

The invention provides a beneficial alternative to using currentlyavailable cross-linking reagents to produce carrier polypeptide-targetpolypeptide conjugates. The methods provided herein include reacting afirst chemically reactive unnatural amino acid residue, which has beendirectly incorporated into a carrier polypeptide variant, with a secondchemically reactive unnatural amino acid residue, which has beendirectly incorporated into a target polypeptide variant, to produce astable, well-defined conjugate. Such conjugates can optionally find useas therapeutic agents with novel or improved biological properties,reduced toxicity, enhanced activities, and/or increased half-life.Advantageously, these methods can maximize yields and minimize costs bypermitting the consistent production of homogenous populations ofconjugates, e.g., that each comprise identical stoichiometries andligation sites.

As will be apparent from the description herein, various embodiments ofthe invention can comprise any of a wide variety of carrier polypeptidevariants that include a first reactive unnatural amino acid and any of awide variety of target polypeptide variants that include a secondreactive unnatural amino acid, wherein the first and second unnaturalamino acids are reacted to form the stable, covalently linked carrierpolypeptide-target polypeptide conjugate. The carrier polypeptide and/ortarget polypeptide can optionally be therapeutic, immunogenic, derivedfrom a foreign organism, a self-protein, synthetic, etc. Particularcarrier polypeptides of interest are described in detail hereinbelow.Target polypeptides that find use with the invention include, but arenot limited to, such small therapeutic peptides as TSP-1, ABT-510,GLP-1, Exedin-4, peptide derivatives of the preceding, and others, whichare described in further detail elsewhere herein.

In certain embodiments described herein, the unnatural amino acids canbe site-specifically incorporated into a carrier or target polypeptidevariant with high efficiency and high fidelity using orthogonaltRNA/aminoacyl-tRNA synthetase pairs, e.g., in methylotrophic yeast suchas P. pastoris, P. methanolica, P. angusta (also known as Hansenulapolymorpha), Candida boidinii, and Torulopsis spp. Methylotrophic yeastare attractive candidates for use as recombinant expression systems forheterologous, therapeutically useful proteins (Lin-Cereghino et al.(2000) “Heterologous protein expression in the methylotrophic yeastPichia pastoris.” FEMS Microbiol Rev 24: 45-66; International PatentApplication Number PCT/US2008/013568, filed Dec. 10, 2008, entitled, “InVivo Unnatural Amino Acid Expression in the Methylotrophic Yeast PichiaPastoris”; and US Patent Application Publication 2009/0197339, filedDec. 10, 2008, entitled, “In Vivo Unnatural Amino Acid Expression in theMethylotrophic Yeast Pichia Pastoris”. The eukaryotic subcellularorganization of methylotrophic yeast enables them to carry out many ofthe posttranslational folding, processing and modification eventsrequired to synthesize biologically active carrier polypeptides and/ortarget polypeptides derived from mammals. Unlike proteins expressed inS. cerevisiae, proteins produced by methylotrophic yeast such as P.pastoris, P. methanolica, P. angusta (also known as Hansenulapolymorpha), Candida boidinii, and Torulopsis spp., are less likely tocontain high-mannose glycan structures that can hamper downstreamprocessing of heterologously expressed glycoproteins. In addition,carrier and/or target polypeptides synthesized in methylotrophic yeastare advantageously free of pyrogenic and antigenic compounds oftencharacteristic of proteins expressed in E. coli. Most significantly,methylotrophic yeast expression systems are particularly useful forlarge-scale synthesis. For example, orthogonal translation systems inmethylotrophic yeast can permit the expression of recombinant carrierand/or target polypeptides comprising unnatural amino acids at levels10- to 100-fold higher than in S. cerevisiae, bacterial, insect, ormammalian systems. In addition, methylotrophic yeast can be easilycultured in a simple, defined salt medium, eliminating the need for theexpensive media supplements and equipment that are required forbaculovirus expression systems. Further details regarding the use oforthogonal translation systems in methylotrophic yeast can be found inInternational Patent Application Number PCT/US2008/013568, filed Dec.10, 2008, entitled, “In Vivo Unnatural Amino Acid Expression in theMethylotrophic Yeast Pichia Pastoris” and US Patent ApplicationPublication 2009/0197339, filed Dec. 10, 2008, entitled, “In VivoUnnatural Amino Acid Expression in the Methylotrophic Yeast PichiaPastoris”.

First and second unnatural amino acids can be directly incorporated intocarrier and target polypeptides, respectively, using any of a number ofmethods known in the art. While many embodiments utilize orthogonaltranslation systems, e.g., in P. pastoris (see below) or othermethylotrophic yeast (e.g., P. methanolica, P. angusta (or Hansenulapolymorpha), Candida boidinii, and Torulopsis spp), as the route ofdirect incorporation of the unnatural amino acids, other directincorporation methods (e.g., in vitro translation systems, solid-phasesynthesis, etc.) can also optionally be used. It will be appreciatedthat in typical embodiments herein, an unnatural amino acid isincorporated into a carrier and/or target polypeptide duringconstruction of the polypeptide and is not added via post-translationalchemical derivatization.

In one illustrative example, the invention provides methods andcompositions useful for the production of an HSA-ABT-510 conjugate.ABT-510 is an antiangiogenic peptide mimetic derived from humanthrombospondin that exhibits potent antitumor activity in humans, butwhich suffers from rapid clearance by the kidneys when administeredintravenously (Hoekstra et al. (2005) “Phase I safety, pharmacokinetic,and pharmacodynamic study of the thrombospondin-1-mimetic angiogenesisinhibitor ABT-510 in patients with advanced cancer.” J Clin Oncol 23:5188-5197; Yang et al. (2007) “Thrombospondin-1 peptide ABT-510 combinedwith valproic acid is an effective antiangiogenesis strategy inneuroblastoma.” Cancer Res 67: 1716-1724; Reiher et al. (2002)“Inhibition of tumor growth by systemic treatment with thrombospondin-1peptide mimetics.” Int J Cancer 98: 682-689). HSA is awell-characterized soluble serum protein with a half-life of 19 days,which functions primarily as a carrier protein for steroids, fattyacids, and thyroid hormones. HSA is described, for example, in entry103600 in the Online Mendelian Inheritance in Man database (see alsoGenBank Accession No. AAA98797.1). A p-acetylphenylalanine residue,which has been incorporated into an HSA variant at amino acid position37, and a ε-(2-(aminooxy)acetyl)-L-lysine residue, which has beenincorporated into an ABT-510 variant at amino acid position 6, react viaselective oxime ligation to produce an HSA-ABT-510 conjugate.Conjugation to HSA can prolong the conjugated peptide's half-life, thusincreasing its therapeutic utility. A p-acetylphenylalanine residue,which has been incorporated into an HSA variant at amino acid position37, and a ε-(2-(aminooxy)acetyl)-L-lysine residue, which has beenincorporated into an ABT-510 variant at amino acid position 1 can alsobe reacted, via oxime ligation, to produce a useful conjugate.

The detailed description is organized to first elaborate the variouscarrier polypeptides and target polypeptides that can be chemicallyligated using the methods and compositions provided by the invention.Next, details regarding methods of producing carrier polypeptidevariants and/or target polypeptide variants comprising first and secondreactive unnatural amino acids, respectively, are described. Chemicalcoupling reactions that can be used to conjugate a carrier polypeptidevariant to a target polypeptide variant are then elaborated.Pharmaceutical compositions comprising conjugates of the invention anddetails regarding their administration are described thereafter. Lastly,kits comprising conjugates of the invention and/or methods of theirproduction are described.

Carrier Polypeptides and Target Polypeptides of Interest

As described above, the invention provides methods and compositions forthe production of carrier polypeptide-target polypeptide conjugateswherein first and second unnatural amino acid residues with uniquechemical functionalities that have been directly incorporated intocarrier polypeptides and target polypeptides, respectively, are reactedto produce a homogenous population of defined conjugates. The reactiveunnatural amino acids present in carrier and target polypeptide variantscan optionally be in any location within the polypeptides. Placement ofthe unnatural amino acids in the carrier and target polypeptide variantsis optionally chosen based on, e.g., whether placement in that locationwould change, e.g., the conformations, biological activities,pharmacologically activities, or other relevant properties, of thecarrier and/or target polypeptide variants vs. the “natural”polypeptides from which they are derived. Another criterion by which thelocations of chemically reactive unnatural amino acids in carrier andtarget polypeptides are optionally selected is whether the locationsallow the reactive unnatural amino acids to be accessible (e.g., can thetwo unnatural amino acids participate in a ligation reaction to producea stable conjugate), etc.

The first and second amino acids that are incorporated into the carrierand target polypeptide variants, respectively, can be conservative ornon-conservative replacements (as compared to the corresponding naturalamino acids in the “natural” carrier and target polypeptides thatcomprise no unnatural amino acids). A chemically reactive unnaturalamino acid in a carrier polypeptide and/or a target polypeptide canoptionally replace a natural amino acid in the carrier and/or targetpolypeptide's primary sequence. Alternatively or additionally, one (ormore) reactive unnatural amino acids can be added to the primarysequence of a carrier or target polypeptide.

The carrier and target polypeptides that include the first and secondreactive unnatural amino acids, respectively, can be constructed in anyof a number of methods that entail the direct incorporation of anunnatural amino acid into a growing polypeptide chain. While manyembodiments utilize orthogonal translation in, e.g., P. pastoris (seebelow) or other methylotrophic yeast such as P. methanolica, P. angusta(also known as Hansenula polymorpha), Candida boidinii, and Torulopsisspp., as the route of direct incorporation of the unnatural amino acids,other direct incorporation methods (e.g., in vitro translation systems,solid-phase synthesis, etc.) can also optionally be used, or the methodscan be used in combination. However, advantages to expression inmethylotrophic yeast include their ease of genetic manipulation, theireconomy of recombinant protein production, and their abilities toperform complex posttranslational modifications typically associatedwith eukaryotic proteins. These and other advantages of recombinantprotein expression in methylotrophic yeast are explained in furtherdetail below.

In certain embodiments, a carrier polypeptide can be conjugated to morethan one target polypeptide, e.g., using one or more of the ligationchemistries described herein, to produce, e.g., a therapeutically usefulcarrier-target conjugate such as a multiple antigen peptide (MAP).Conversely, a target polypeptide can optionally be conjugated tomultiple carrier polypeptides.

HSA and ABT-510 were chosen as the carrier polypeptide and targetpolypeptide, respectively, to illustrate aspects of the currentinvention. HSA is a well-characterized serum protein with a serumhalf-life of 19 days, and ABT-510, a peptide derivative of the naturalangiogenic inhibitor thrombospondin-1, exhibits potent antitumoractivity in humans but suffers from rapid clearance by the kidneys whenadministered intravenously (Hoekstra et al. (2005) “Phase I safety,pharmacokinetic, and pharmacodynamic study of thethrombospondin-1-mimetic angiogenesis inhibitor ABT-510 in patients withadvanced cancer.” J Clin Oncol 23: 5188-5197; Yang et al. (2007)“Thrombospondin-1 peptide ABT-510 combined with valproic acid is aneffective antiangiogenesis strategy in neuroblastoma.” Cancer Res 67:1716-1724; Reiher et al. (2002) “Inhibition of tumor growth by systemictreatment with thrombospondin-1 peptide mimetics.” Int J Cancer 98:682-689). As described in the Example below, a p-acetylphenylalaine thathas been incorporated into an HSA or an HSA variant can be reacted witha ε-(2-(aminooxy)acetyl)-L-lysine that has been incorporated into anABT-510 or ABT-510 variant, e.g., via oxime ligation. It will beappreciated that the illustrations in the Example below are not the onlyembodiments of the invention. As will be apparent from the descriptionherein, various embodiments of the invention can comprise any of a widevariety of carrier polypeptide variants that include a first reactiveunnatural amino acid and any of a wide variety of target polypeptidevariants that include a second reactive unnatural amino acid, whereinthe first and second unnatural amino acids are reacted to form a stable,covalently linked carrier polypeptide-target polypeptide conjugate.Thus, in various embodiments, the carrier polypeptide and/or targetpolypeptide can optionally be therapeutic, immunogenic, derived from aforeign organism, a self-protein, synthetic, etc. Particular carrier andtarget polypeptides of interest are described below.

Carrier Polypeptides of Interest

As described elsewhere herein, a carrier protein can be beneficiallylinked to a target polypeptide, e.g., a therapeutic peptide such asABT-510, using the methods provided be the invention, in order to, e.g.,promote the target polypeptide's delivery into cells, to increase itsserum half-life, to maintain stabilize its activity, to prevent itsaggregation following administration to a subject, etc. Preferredcharacteristics of a carrier protein can include high solubility and along half-life; however, it is not intended that a carrier polypeptideused in the invention be limited by possessing or not possessing anyparticular biological activity.

The carrier polypeptide can optionally comprise, be, or be homologousto, e.g., an antibody (e.g., an OKT3 antibody, a HER2 antibody, etc.),an antibody fragment (e.g., an scFv, an Fab, an Fc, etc.), an albumin, aserum albumin, a bovine serum albumin, an ovalbumin, a c-reactiveprotein, a conalbumin, a lactalbumin, a keyhole limpet hemocyanin (KLH),an ion carrier protein, an acyl carrier protein, a signal transducingadaptor protein, an androgen-binding protein, a calcium-binding protein,a calmodulin-binding protein, a ceruloplasmin, a cholesterol estertransfer protein, an f-box protein, a fatty acid-binding proteins, afollistatin, a follistatin-related protein, a GTP-binding protein, aninsulin-like growth factor binding protein, an iron-binding protein, alatent TGF-beta binding protein, a light-harvesting protein complex, alymphocyte antigen, a membrane transport protein, a neurophysin, aperiplasmic binding protein, a phosphate-binding protein, aphosphatidylethanolamine binding protein, a phospholipid transferprotein, a retinol-binding protein, an RNA-binding protein, an s-phasekinase-associated protein, a sex hormone-binding globulin, athyroxine-binding protein, a transcobalamin, a transcortin, atransferrin-binding protein, or a vitamin D-binding protein.

However, it will be appreciated that the methods and compositions of theinvention are not limited to the polypeptides listed above. A carrierpolypeptide-target polypeptide conjugate of the invention can optionallycomprise any carrier polypeptide well known in the art

Target Polypeptides of Interest

Target polypeptides can include any polypeptide of interest whosehalf-life, pharmacological activity, biological activity,bioavailablility can be stabilized or improved by the chemical ligationof the target polypeptide to a carrier protein. Target polypeptides canoptionally be therapeutic, immunogenic, derived from a foreign organism,a self-protein, synthetic, etc. In particularly useful embodiments, atarget polypeptide can be a small therapeutic peptide (see below).However, it is not intended that a target polypeptide be limited bypossessing or not possessing any particular biological activity. Inembodiments herein, a target polypeptide can comprise, be a variant of,or be homologous to, e.g., a TSP-1, an ABT-510, a glugacon-likepeptide-1 (GLP-1), a parathyroid hormone (PTH), an Exedin-4, a ribosomeinactivating protein (RIP), an angiostatin, an apoprotein, an atrialnatriuretic factor, an atrial natriuretic polypeptide, an atrialpeptide, a C-X-C chemokine, a T39765, a NAP-2, an ENA-78, a gro-a, agro-b, a gro-c, an IP-10, a GCP-2, a NAP-4, an a PF4, a MIG, acalcitonin, a c-kit ligand, a cytokine, a CC chemokine, a monocytechemoattractant protein-1, a monocyte chemoattractant protein-2, amonocyte chemoattractant protein-3, a monocyte inflammatory protein-1alpha, a monocyte inflammatory protein-1 beta, a RANTES, an I309, anR83915, an R91733, a T58847, a D31065, a T64262, a CD40 ligand, acomplement inhibitor, a cytokine, an epithelial neutrophil activatingpeptide-78, a GROΥ, a MGSA, a GROβ, a GROγ, a MIP1-α, a MIP1-β, anMCP-1, an epithelial neutrophil activating peptide, an erythropoietin(EPO), an exfoliating toxin, a fibroblast growth factor (FGF), an FGF21,a G-CSF, a gonadotropin, a growth factor, a Hirudin, an LFA-1, a humaninsulin, a human insulin-like growth factor (hIGF), an hIGF-I, anhIGF-II, a human interferon, an IFN-α, an IFN-β, an IFN-γ, aninterleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, anIL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a keratinocytegrowth factor (KGF), a leukemia inhibitory factor, a neurturin, a PDGF,a peptide hormone, a pleiotropin, a pyrogenic exotoxin A, a pyrogenicexotoxin B, a pyrogenic exotoxin C, a relaxin, a somatostatin, asuperoxide dismutase, a thymosin alpha 1, a human tumor necrosis factor(hTNF), a human tumor necrosis factor alpha, a human tumor necrosisfactor beta, a Ras, a Tat, an inflammatory molecule, a signaltransduction molecule, a bovine pancreatic trypsin inhibitor (BPTI), ora BP320 antigen. Again, it will be appreciated that the methods andcompositions of the invention are not limited to those targetpolypeptides listed above.

Methods of Producing Carrier Polypeptide Variants and/or TargetPolypeptide Variants

The carrier polypeptide variants and/or target polypeptide variants,e.g., that are to be reacted to produce the conjugates of the invention,can be produced using any of a variety of methods that entail the directincorporation of an unnatural amino acid into a growing polypeptidechain. Thus, while the description and the examples primarily focus onthe use of orthogonal translation systems in methylotrophic yeast, e.g.,P. pastoris (see below), P. methanolica, P. angusta (also known asHansenula polymorpha), Candida boidinii, and Torulopsis spp., toincorporate chemically reactive first and second unnatural amino acidsinto carrier polypeptide variants and target polypeptide variants,respectively, other orthogonal translation systems and/or othernon-orthogonal direct incorporation methods can optionally be used toproduce the target and carrier polypeptide variants that comprise suchunnatural amino acids. In several embodiments, a chemically reactiveunnatural amino acid can be incorporated into a carrier polypeptidevariant or a target polypeptide variant, e.g., a small therapeuticpeptide, while the polypeptide is being made, e.g., during translationusing O-tRNA/O-RS pairs, during in vitro synthesis usingchemico-synthetic methods, etc. Therefore, while particular methods ofconstructing carrier and/or target polypeptide variants that compriseunnatural amino acids are detailed herein, e.g., using orthogonaltranslation systems in methylotrophic yeast, they such should notnecessarily be taken as limiting. Other methods in which unnatural aminoacids are directly incorporated into carrier and/or polypeptides duringprimary assembly are also included herein in the many embodiments.

Following incorporation of the reactive first and second unnatural aminoacids into a carrier polypeptide and target polypeptide, respectively,e.g., using any of the methods detailed hereinbelow, the first unnaturalamino acid present in the carrier polypeptide variant can be reactedwith the second unnatural amino acid present in the target polypeptidevariant, e.g., via any one of the ligation chemistries describedelsewhere herein, to form a stable, defined conjugate.

It will be appreciated that genetic incorporation of unnatural aminoacids into carrier polypeptides and target polypeptides (e.g., throughorthogonal translation systems such as those described and referenced toherein) prior to the formation of stable carrier polypeptide-targetpolypeptide conjugates can, in some embodiments, offer benefits overgeneration of carrier polypeptide-target polypeptide conjugates usingmethods that are currently available. For example, one currentmethodology for conjugation of target polypeptides to the carrier HSAinvolves exploitation of its single free cysteine (residue 34) viamaleimide linkages. This strategy has several drawbacks, including theantigenicity of maleimide and the frequent modification of cysteine 34by endogenous nitric oxide, free cysteine, glutathione, or sugars, whichcan lead to variable coupling efficiency.

The genetic incorporation of unnatural amino acids into carrierpolypeptides and/or target polypeptides using an in vivo orthogonaltranslation system can produce biologically and/or pharmacologicallyactive carrier polypeptide variants and/or target polypeptide variants,but with the added active/functional groups introduced via the directincorporation of unnatural amino acids, which functional groups can bereacted to produce stable carrier-target polypeptide conjugates.Furthermore, use of the novel biotechnological tool of in vivoincorporation of unnatural amino acids can help produce the propernative conformation of carrier and/or target polypeptides that compriseunnatural amino acids in high yields at low cost. In addition, thereactivities of the first and second unnatural amino acids that areincorporated into the carrier and target polypeptide variants,respectively, can be deliberately chosen based on such criteria asmolecular linker length, reaction conditions, whether the resultinglinker is cleavable, etc.

In contrast, total synthesis of carrier or target polypeptide variantswith unnatural amino acids using other in vitro methods such assolid-phase peptide synthesis can, in some embodiments, be targeted toshorter molecules (e.g., ˜60-100 amino acids) as well as producingdenatured proteins at a lower yield.

In certain embodiments, orthogonal translation systems in methylotrophicyeast are used to produce a carrier polypeptide variant, e.g., HSA, thatcomprises an unnatural amino acid, e.g., p-acetylphenylalanine, that canbe reacted with a second unnatural amino acid, e.g.,ε-(2-(aminooxy)acetyl)-L-lysine, in a target polypeptide variant, e.g.,TSP-1 or ABT-510, to form a stable carrier polypeptide-targetpolypeptide conjugate. The use of O-RS/O-tRNA pairs in methylotrophicyeast such as Pichia pastoris (see below), P. methanolica, P. angusta(also known as Hansenula polymorpha), Candida boidinii, and Torulopsisspp. is characterized by several beneficial advantages over otherorthogonal systems, e.g., those in E. coli, S. cerevisiae, or mammaliancells, for the production of such proteins. As described furtherhereinbelow, their ease of genetic manipulation, their economy ofrecombinant protein production, and their abilities to perform theposttranslational modifications typically associated with eukaryoticproteins make methylotrophic yeast an advantageous system for theexpression of heterologous proteins, e.g., carrier polypeptide variantsand/or target polypeptide variants, that comprise unnatural amino acids.

Orthogonal tRNA/Orthogonal Aminoacyl t-RNA Synthetase Technology

In certain embodiments, chemically reactive first and second unnaturalamino acids that can participate in chemical ligation reactions, e.g.,those described elsewhere herein, are incorporated into carrierpolypeptides and/or target polypeptides, respectively, prior to thechemical ligation reaction via orthogonal tRNA (O-tRNA)/aminoacyl-tRNAsynthetase (O-RS) systems. Thus, an understanding of the compositionsand methods provided by the invention is further developed through anunderstanding of the activities associated with O-tRNA/O-RS pairs. Ingeneral, in order to add unnatural amino acids to the genetic code, neworthogonal pairs comprising an aminoacyl-tRNA synthetase and a suitabletRNA are needed that can function efficiently in the host translationalmachinery, but that are “orthogonal” to the translation system at issue.Thus, the orthogonal moieties function independently of the synthetasesand tRNAs endogenous to the translation system. Desired characteristicsof the orthogonal pair include a tRNA that decodes or recognizes only aspecific codon, such as a selector codon, e.g., an amber stop codon,that is not decoded by any endogenous tRNA, and an aminoacyl-tRNAsynthetase that preferentially aminoacylates, or “charges” its cognatetRNA with only one specific unnatural amino acid. The O-tRNA is also nottypically aminoacylated, or is poorly aminoacylated, i.e., charged, byendogenous synthetases.

The general principles of orthogonal translation systems that aresuitable for making proteins that comprise one or more unnatural aminoacid in the invention are known in the art, as are the general methodsfor producing orthogonal translation systems. For example, seeInternational Publication Numbers: WO 2002/086075, entitled “METHODS ANDCOMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNASYNTHETASE PAIRS”; WO 2002/085923, entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS”; WO 2004/094593, entitled “EXPANDING THEEUKARYOTIC GENETIC CODE”; WO 2005/019415, filed Jul. 7, 2004; WO2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004; WO2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATIONCOMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS”; WO2007/103490, filed Mar. 7, 2007, entitled “SYSTEMS FOR THE EXPRESSION OFORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS”, andInternational Patent Application Number PCT/US2008/013568, filed Dec.10, 2008, entitled, “In Vivo Unnatural Amino Acid Expression in theMethylotrophic Yeast Pichia Pastoris”. See also, e.g., Liu et al. (2007)“Genetic incorporation of unnatural amino acids into proteins inmammalian cells” Nat Methods 4: 239-244; Int'l ApplicationPCT/US2008/081868 entitled “A Genetically Encoded Boronate Amino Acid,”filed Oct. 30, 2008; WO2007/047301 entitled “Selective PosttranslationalModification of Phage-Displayed Polypeptides,” filed Oct. 11, 2006; andWO2006/110182 entitled “Orthogonal Translation Components for the invivo Incorporation of Unnatural Amino Acids,” filed Oct. 27, 2005. Eachof the aforementioned applications is incorporated herein by referencein its entirety. For discussion of orthogonal translation systems thatincorporate unnatural amino acids, and methods for their production anduse, see also, Wang and Schultz (2005) “Expanding the Genetic Code”Angewandte Chemie Int Ed 44: 34-66; Xie and Schultz (2005) “An ExpandingGenetic Code” Methods 36: 227-238; Xie and Schultz (2005) “Adding AminoAcids to the Genetic Repertoire” Curr Opinion in Chemical Biology 9:548-554; Wang et al. (2006) “Expanding the Genetic Code” Annu RevBiophys Biomol Struct 35: 225-249; Deiters et al. (2005) “In vivoincorporation of an alkyne into proteins in Escherichia coli” Bioorganic& Medicinal Chemistry Letters 15: 1521-1524; Chin et al. (2002)“Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichiacoli” J Am Chem Soc 124: 9026-9027; and International Publication No.WO2006/034332, filed on Sep. 20, 2005. The contents of each of thesedocuments are incorporated by reference in its entirety. Additionaldetails of orthogonal translation systems can be found in U.S. Pat. Nos.7,045,337; 7,083,970; 7,238,510; 7,129,333; 7,262,040; 7,183,082;7,199,222; and 7,217,809.

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than the twentygenetically encoded alpha-amino acids. As used herein, a selenocysteineor a pyrrolysine can be incorporated into a carrier polypeptide and/ortarget polypeptide. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids, although unnatural aminoacids can be naturally occurring compounds other than the twentyproteinogenic alpha-amino acids. Unnatural amino acids finding use withthe invention include an p-(propargyloxy)phenylalanine,p-methoxyphenylalanine, dansylalanine, DMNB-serine, O-methyl-L-tyrosine,an L-3-(2-naphthyl)alanine, an O-4-allyl-L-tyrosine, anO-propargyl-L-tyrosine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acetyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, anL-phosphoserine, a phosphonoserine, a phosphonotyrosine, ap-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine,an unnatural analogue of a tyrosine amino acid; an unnatural analogue ofa glutamine amino acid; an unnatural analogue of a phenylalanine aminoacid; an unnatural analogue of a serine amino acid; an unnaturalanalogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano,halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol,sulfonyl, sulfo, seleno, ester, thioacid, borate, boronate, phospho,phosphono, heterocyclic, enone, imine, aldehyde, alkoxyamine,hydroxylamine, keto, or amino substituted amino acid, or any combinationthereof; an amino acid with a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; an amino acid with anovel functional group; an amino acid that covalently or noncovalentlyinteracts with another molecule; a metal binding amino acid; ametal-containing amino acid; a radioactive amino acid; a photocagedand/or photoisomerizable amino acid; a biotin or biotin-analoguecontaining amino acid; a glycosylated or carbohydrate modified aminoacid; a keto containing amino acid; amino acids comprising polyethyleneglycol or polyether; a heavy atom substituted amino acid; a chemicallycleavable or photocleavable amino acid; an amino acid with an elongatedside chain; an amino acid containing a toxic group; a sugar substitutedamino acid, e.g., a sugar substituted serine or the like; acarbon-linked sugar-containing amino acid; a sugar-substituted cysteine;a redox-active amino acid; an α-hydroxy containing acid; an amino thioacid containing amino acid; an α,α disubstituted amino acid; a β-aminoacid; sulfotyrosine, 4-borono-phenylalanine, an aminooxy amino acid, anaminooxy lysine, an aminooxy ornithine, an aminooxy tyrosine, or acyclic amino acid other than proline.

Other unnatural amino acids that can be incorporated into, e.g., targetand/or carrier polypeptides, include, but are not limited to, unnaturalamino acids comprising any one or more of the following functionalgroups: an aldehyde moiety, a keto moiety, a beta-diketo moiety, analkoxyamine moiety, an acyl hydrazide moiety, a dehydroalanine moiety, athioester moiety, an ester moiety, a boronate moiety, an azide moiety,an acetylenic moiety, an olefinic moiety, a vicinal thiol amine moiety,and the like. An unnatural amino acid present in a target or carrierpolypeptide that comprises such functional groups can react with asecond unnatural amino acid, e.g., present in a carrier or targetpolypeptide, respectively, that comprises a reactive nucleophile and/orelectrophile, e.g., a keto moiety, an aldehyde moiety, an alkoxyaminemoiety, an acylhydrazide moiety, an azide moiety, an alkyne moiety, anolefinic moiety, an amino moiety, a thiol moiety, an aminophenol moiety,an iodophenyl moiety, or the like.

Orthogonal Translation Systems

Orthogonal translation systems generally comprise cells, e.g.,prokaryotic cells such as E. coli; and eukaryotic cells such as S.cerevisiae, mammalian cells, and methylotrophic yeast cells (e.g., P.pastoris, P. methanolica, P. angusta (also known as Hansenulapolymorpha), Candida boidinii, and Torulopsis spp.) etc., that includean orthogonal tRNA (O-tRNA), an orthogonal aminoacyl tRNA synthetase(O-RS), and an unnatural amino acid, where the O-RS aminoacylates theO-tRNA with the unnatural amino acid. An orthogonal pair can include anO-tRNA, e.g., a suppressor tRNA, a frameshift tRNA, or the like, and acognate O-RS. Orthogonal systems that can be used to produce the carrierpolypeptides and/or target polypeptide variants herein, which typicallyinclude O-tRNA/O-RS pairs, can comprise a cell or a cell-freeenvironment.

In general, when an orthogonal pair recognizes a selector codon andloads an amino acid in response to the selector codon, the orthogonalpair is said to “suppress” the selector codon. That is, a selector codonthat is not recognized by the translation system's endogenous machineryis not ordinarily charged, which results in blocking production of apolypeptide that would otherwise be translated from the nucleic acid. Inan orthogonal pair system, the O-RS aminoacylates the O-tRNA with aspecific unnatural amino acid, e.g., p-acetylphenylalanine, as used inthe Example herein. The charged O-tRNA recognizes the selector codon andsuppresses the translational block caused by the selector codon, e.g.,producing an HSA variant that comprises a p-acetylphenylalanine, e.g.,at amino acid position 37.

The translation system uses the O-tRNA/O-RS pair to incorporate anunnatural amino acid into a growing polypeptide chain, e.g., via apolynucleotide that encodes a polypeptide of interest (such as a carrierpolypeptide and/or a target polypeptide), where the polynucleotidecomprises a selector codon that is recognized by the O-tRNA. In certainsystems, the cell can include one or more additional O-tRNA/O-RS pairs,where an additional O-tRNA is loaded by an additional O-RS with adifferent unnatural amino acid. For example, one of the O-tRNAs canrecognize a four base codon and the other O-tRNA can recognize a stopcodon. Alternately, multiple different stop codons, multiple differentfour base codons, multiple different rare codons and/or multipledifferent non-coding codons can be used in the same coding nucleic acid.Thus, a single polypeptide, e.g., carrier polypeptide and/or targetpolypeptide variant, can comprise multiple unnatural amino acids.Alternatively or additionally, different carrier and/or targetpolypeptide or polypeptide variants created in the system can comprisedifferent unnatural amino acids. For further details regarding availableO-RS/O-tRNA cognate pairs and their use, see, e.g., the references notedelsewhere herein.

Thus, some translational systems can comprise multiple O-tRNA/O-RSpairs, which allow incorporation of more than one unnatural amino acidinto a carrier polypeptide variant and/or a target polypeptide variant.For example, the translation system can further include an additionaldifferent O-tRNA/O-RS pair and a second unnatural amino acid, where thisadditional O-tRNA recognizes a second selector codon and this additionalO-RS preferentially aminoacylates the O-tRNA with the second unnaturalamino acid. For example, a cell that includes an O-tRNA/O-RS pair, wherethe O-tRNA recognizes, e.g., an amber selector codon, can furthercomprise a second orthogonal pair, where the second O-tRNA recognizes adifferent selector codon, e.g., an opal codon, an ochre codon, afour-base codon, a rare codon, a non-coding codon, or the like. In somesystems, the different orthogonal pairs are derived from differentsources, which can facilitate recognition of different selector codons.

Certain translation systems can comprise a cell, such as an E. colicell, a mammalian cell, an S. cerevisiae cell, or a methylotrophic yeastcell (e.g., a P. pastoris cell, a P. methanolica cell, a P. angusta (orHansenula polymorpha) cell, a Candida boidinii cell, or a Torulopsisspp. cell) that includes an orthogonal tRNA (O-tRNA), an orthogonalaminoacyl-tRNA synthetase (O-RS), an unnatural amino acid, and a nucleicacid that comprises a polynucleotide that encodes a polypeptide ofinterest, e.g., a carrier polypeptide variant and/or a targetpolypeptide variant, where the polynucleotide comprises the selectorcodon that is recognized by the O-tRNA. Although orthogonal translationsystems can utilize cultured cells to produce proteins having unnaturalamino acids, it is not intended that orthogonal translation systems usedherein require an intact, viable cell. For example, an orthogonaltranslation system can utilize a cell-free system in the presence of acell extract. Indeed, the use of cell free, in vitrotranscription/translation systems for protein production is a wellestablished technique. Adaptation of these in vitro systems to produceproteins having unnatural amino acids using orthogonal translationsystem components described herein is well within the scope of theinvention.

The O-tRNA and/or the O-RS can be naturally occurring or can be, e.g.,derived by mutation of a naturally occurring tRNA and/or RS, e.g., bygenerating libraries of tRNAs and/or libraries of RSs, from any of avariety of organisms and/or by using any of a variety of availablemutation strategies. For example, one strategy for producing anorthogonal tRNA/aminoacyl-tRNA synthetase pair involves importing atRNA/synthetase pair that is heterologous to the system in which thepair will function from a source, or multiple sources, other than thetranslation system in which the tRNA/synthetase pair will be used. Theproperties of the heterologous synthetase candidate include, e.g., thatit does not charge any host cell tRNA, and the properties of theheterologous tRNA candidate include, e.g., that it is not aminoacylatedby any host cell synthetase. In addition, the heterologous tRNA isorthogonal to all host cell synthetases. A second strategy forgenerating an orthogonal pair involves generating mutant libraries fromwhich to screen and/or select an O-tRNA or O-RS. Such strategies canalso be combined.

Orthogonal tRNA (O-tRNA)

An orthogonal tRNA (O-tRNA) desirably mediates incorporation of anunnatural amino acid into a polypeptide encoded by a polynucleotide thatcomprises a selector codon recognized by the O-tRNA, e.g., in vivo or invitro.

Thus compositions comprising an O-tRNA can further include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates the O-tRNA with an unnatural amino acid. Such compositionsincluding an O-tRNA can further include a translation system, e.g., invitro or in vivo. A nucleic acid that comprises a polynucleotide thatencodes a polypeptide of interest, where the polynucleotide comprises aselector codon that is recognized by the O-tRNA, or a combination of oneor more of these can also be present in the cell.

Methods for producing a recombinant orthogonal tRNA and screening itsefficiency with respect to incorporating an unnatural amino acid into apolypeptide in response to a selector codon can be found in, e.g.,International Application Publications WO 2002/086075, entitled “METHODSAND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNASYNTHETASE PAIRS”; WO 2004/094593, entitled “EXPANDING THE EUKARYOTICGENETIC CODE”; and WO 2005/019415, filed Jul. 7, 2004. See also Forsteret al. (2003) “Programming peptidomimetic synthetases by translatinggenetic codes designed de novo.” Proc Natl Acad Sci USA 100: 6353-6357;and Feng et al. (2003) “Expanding tRNA recognition of a tRNA synthetaseby a single amino acid change.” Proc Natl Acad Sci USA 100:5676-5681.Additional details can be found in U.S. Pat. Nos. 7,045,337; 7,083,970;7,238,510; 7,129,333; 7,262,040; 7,183,082; 7,199,222; and 7,217,809.

Orthogonal Aminoacyl-tRNA Synthetase (O-RS)

The O-RS of systems used to produce, e.g., carrier and/or targetpolypeptide variants that comprise unnatural amino acids, preferentiallyaminoacylates an O-tRNA with an unnatural amino acid either in vitro orin vivo. The O-RS can be provided to the translation system by apolypeptide that includes an O-RS and/or by a polynucleotide thatencodes an O-RS or a portion thereof.

General details for producing an O-RS, assaying its aminoacylationefficiency, and/or altering its substrate specificity can be found inInternal Publication Number WO 2002/086075, entitled “METHODS ANDCOMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNASYNTHETASE PAIRS”; and WO 2004/094593, entitled “EXPANDING THEEUKARYOTIC GENETIC CODE.” See also, Wang and Schultz (2005) “Expandingthe Genetic Code.” Angewandte Chemie Int Ed 44: 34-66; and Hoben andSoll (1985) Methods Enzymol 113: 55-59, the contents of which areincorporated by reference in their entirety. Additional detailsconcerning such systems can be found in U.S. Pat. Nos. 7,045,337;7,083,970; 7,238,510; 7,129,333; 7,262,040; 7,183,082; 7,199,222; and7,217,809

Source and Host Organisms

The orthogonal translational components (O-tRNA and O-RS) that canoptionally be used to create, e.g., the carrier polypeptide variants andtarget polypeptide variants comprising unnatural amino acids that reactto form the stable carrier-target conjugates of the invention, can bederived from any organism, or a combination of organisms, for use in ahost translation system from any other species, with the caveat that theO-tRNA/O-RS components and the host system work in an orthogonal manner.It is not a requirement that the O-tRNA and the O-RS from an orthogonalpair be derived from the same organism. For example, the orthogonalcomponents can be derived from archaebacterial genes for use in aeubacterial host system.

Furthermore, the orthogonal O-tRNA can be derived from anarchaebacterium, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcusmaripaludis, Methanopyrus kandleri, Methanosarcina mazei (Mm),Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss),Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium,or the like, or a eubacterium, such as Escherichia coli, Thermusthermophilus, Bacillus subtilis, Bacillus stearothermphilus, or thelike, while the orthogonal O-RS can be derived from an organism orcombination of organisms, e.g., an archaebacterium, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyruskandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcusabyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasmaacidophilum, Thermoplasma volcanium, or the like, or a eubacterium, suchas Escherichia coli, Thermus thermophilus, Bacillus subtilis, Bacillusstearothermphilus, or the like. In other systems, eukaryotic sources,e.g., plants, algae, protists, fungi, yeasts, animals, e.g., mammals,insects, arthropods, or the like can also be used as sources of O-tRNAsand O-RSs. Furthermore, the individual components of an O-tRNA/O-RS paircan be derived from the same organism or different organisms.

The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or screened in vivoor in vitro and/or used in a cell, e.g., a eubacterial cell, to producea polypeptide with an unnatural amino acid. The eubacterial cell used isnot limited and can include, for example, Escherichia coli, Thermusthermophilus, Bacillus subtilis, Bacillus stearothermphilus, or thelike.

Selector Codons

Various selector codons expand the genetic codon framework of proteinbiosynthetic machinery. For example, a selector codon can include, e.g.,a unique three base codon, a nonsense codon, such as a stop codon, e.g.,an amber codon (UAG), or an opal codon (UGA), an unnatural codon, atleast a four base codon, a rare codon, or the like. A number of selectorcodons can be introduced into a desired gene, e.g., one or more, two ormore, more than three, etc. Conventional site-directed mutagenesis canbe used to introduce the selector codon at the site of interest in apolynucleotide encoding a polypeptide of interest (e.g., a self antigenof a subject, etc.). See, e.g., Sayers et al. (1988) “5′, 3′ Exonucleasein phosphorothioate-based oligonucleotide-directed mutagenesis.” NuclAcid Res 16: 791-802. By using different selector codons, multipleorthogonal tRNA/synthetase pairs can be used that allow the simultaneoussite-specific incorporation of multiple unnatural amino acids e.g.,including at least one unnatural amino acid, using these differentselector codons.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon AGG has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al. (1993) “In vitro protein engineering usingsynthetic tRNA^(Ala) with different anticodons.” Biochemistry 32:7939-7945. In such case, the synthetic tRNA competes with the naturallyoccurring tRNA^(Arg), which exists as a minor species in Escherichiacoli. In addition, some organisms do not use all triplet codons. Anunassigned codon AGA in Micrococcus luteus has been utilized forinsertion of amino acids in an in vitro transcription/translationextract. See, e.g., Kowal and Oliver (1997) “Exploiting unassignedcodons in Micrococcus luteus for tRNA-based amino acid mutagenesis” NuclAcid Res 25: 4685-4689.

Selector codons can also comprise extended codons, e.g., four or morebase codons, such as, four, five, six or more base codons. Examples offour base codons include, e.g., AGGA, CUAG, UAGA, CCCU, and the like.Examples of five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA,CUACU, UAGGC and the like. Particular methods of incorporating unnaturalamino acids into proteins, e.g., carrier polypeptides and/or targetpolypeptides described elsewhere herein, can include using extendedcodons based on frameshift suppression. Four or more base codons caninsert, e.g., one or multiple unnatural amino acids, into the sameprotein. In other instances, the anticodon loops can decode, e.g., atleast a four-base codon, at least a five-base codon, or at least asix-base codon or more. Since there are 256 possible four-base codons,multiple unnatural amino acids can be encoded in the same cell using afour or more base codon. See also, Anderson et al. (2002) “Exploring theLimits of Codon and Anticodon Size.” Chemistry and Biology 9: 237-244;Magliery et al. (2001) “Expanding the Genetic Code: Selection ofEfficient Suppressors of Four-base Codons and Identification of “Shifty”Four-base Codons with a Library Approach in Escherichia coli.” J MolBiol 307: 755-769; Ma et al. (1993) “In vitro protein engineering usingsynthetic tRNA^(Ala) with different anticodons.” Biochemistry 32: 7939;Hohsaka et al. (1999) “Efficient Incorporation of Nonnatural Amino Acidswith Large Aromatic Groups into Streptavidin in in Vitro ProteinSynthesizing Systems.” J Am Chem Soc 121: 34-40; and Moore et al. (2000)“Quadruplet Codons: Implications for Code Expansion and theSpecification of Translation Step Size.” J Mol Biol 298: 195-209. Fourbase codons have been used as selector codons in a variety of orthogonalsystems. See, e.g., WO 2005/019415; WO 2005/007870; and WO 2005/07624.See also, Wang and Schultz, (2005) “Expanding the Genetic Code,”Angewandte Chemie Int Ed 44: 34-66.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, such can include a systemthat is lacking a tRNA that recognizes the natural three base codon,and/or a system where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. Descriptions ofunnatural base pairs which can be adapted for use with the methods andcompositions herein include, e.g., Hirao et al. (2002) “An unnaturalbase pair for incorporating amino acid analogues into protein.” NatureBiotechnology 20: 177-182. See also, Wu et al. (2002) “EnzymaticPhosphorylation of Unnatural Nucleosides.” J Am Chem Soc 124:14626-14630.

Expression and Purification of Carrier Polypeptides and/or TargetPolypeptides Comprising Unnatural Amino Acids Using OrthogonalTranslation Systems in Methylotrophic Yeast

In embodiments described in the Example, the carrier polypeptide HSAcomprising a first unnatural amino acid, p-acetylphenylalanine isproduced, e.g., in preparation for conjugation to a target polypeptidecomprising a second unnatural amino acid, using an orthogonaltranslation system in the methylotrophic yeast Pichia Pastoris. Theorthogonal components used in P. pastoris include an O-RS derived fromE. coli tyrosyl tRNA-synthetase, and an O-tRNA, e.g., a mutant tyrosyltRNA_(CUA) amber suppressor derived from E. coli, which function as anorthogonal pair in host P. pastoris cells.

The use of O-RS/O-tRNA pairs in methylotrophic yeast is characterized byseveral beneficial advantages over other orthogonal systems, e.g., E.coli, S. cerevisiae, or mammalian cells, for the production of carrierpolypeptides and/or target polypeptides that comprise unnatural aminoacids. First, the eukaryotic subcellular structure of methylotrophicyeast such as P. pastoris, P. methanolica, P. angusta (Hansenulapolymorpha), Candida boidinii, and Torulopsis spp., permits theincorporation of UAA into proteins that require complexpost-translational modifications, e.g., glycosylation, disulfide bondformation, sulfation, acetylation, prenylation, and proteolyticprocessing, for biological activity, e.g., mammalian proteins such ashuman serum albumin (HSA) and human neutral endopeptidase (NEP). Thus,many proteins that end up as inactive inclusion bodies in bacterialsystems can be produced as biologically active molecules inmethylotrophic yeast. Second, proteins comprising UAA that are expressedin and prepared from in methylotrophs do not contain high concentrationsof pyrogens, e.g., lipopolysaccharides, or antigens, e.g., high-mannoseoligosaccharides, that might hinder the efficacy of proteins expresslydesigned for therapeutic use. Third, many well established techniquesand methods, e.g., gene-targeting, high-frequency DNA transformation,cloning by functional complementation, are available for the geneticmanipulation of foreign genes in methylotrophs (Lin-Cereghino et al,(2002) “Production of recombinant proteins in fermenter cultures of theyeast Pichia pastoris.” Curr Opin Biotechnol 13: 329-332). Theavailability of endogenous inducible promoters and selectable markersadds flexibility to range of proteins comprising UAA that can beproduced by in methylotrophic yeast hosts.

In addition to these advantages, methylotrophs such as P. pastoris, P.methanolica, P. angusta (or Hansenula polymorpha), Candida boidinii, andTorulopsis spp., are also well suited for low-cost, large-scalesynthesis of complex proteins comprising UAA. Methylotrophic yeast areeasily cultured in a simple, defined salt medium, eliminating the needfor the expensive media supplements and costly equipment that arerequired for, e.g., baculovirus expression systems or mammalian tissueculture. In general, methylotrophs can grow to very high cell densities,and, under ideal conditions, methylotrophic yeast can multiply to thepoint where the cell suspension is the consistency of a paste. Theirprolific growth rates allow recombinant methylotrophic yeast strains toproduce carrier and/or target polypeptides comprising UAA at highlevels, e.g., 10- to 100-fold higher level than in S. cerevisiae. Theirease of genetic manipulation, their economy of recombinant proteinproduction, and their abilities to perform the posttranslationalmodifications typically associated with eukaryotic proteins makemethylotrophic yeast an advantageous system for the expression ofheterologous proteins comprising UAA.

The four known genera of methylotrophic yeast, e.g., Hansenula, Pichia,Candida, and Torulopsis, share a common metabolic pathway that enablesthem to use methanol as a sole carbon source. In a transcriptionallyregulated response to methanol induction, several of the enzymes arerapidly synthesized at high levels. Since the promoters controlling theexpression of these genes are among the strongest and most strictlyregulated yeast promoters, methylotrophic yeast have become veryattractive as hosts for the large scale production of recombinantproteins. The cells of these methylotrophic yeast can be grown rapidlyto high densities, and the level of product expression can be regulatedby simple manipulation of the medium. Expression systems have this farbeen developed in P. pastoris, P. methanolica, P. angusta (or Hansenulapolymorpha) and Candida boidinii, and these systems are furtherelaborated in, e.g., Houard et al. (2002)“Engineering ofnon-conventional yeasts for efficient synthesis of macromolecules: themethylotrophic genera.” Biochimie 84: 1089-1093; Gellison (2002)Hansenula Polymorpha: Biology and Applications, 1st Ed., Wiley-VCH, NY;U.S. Pat. No. 6,645,739; Gellisen (2000) “Heterologous proteinproduction in methylotrophic yeasts.” Applied Microbiology andBiotechnology 54: 741-750. Many of these systems are commerciallyavailable, e.g., Hansenula kits from Artes Biotecnology and Pichia kitsfrom Invitrogen, for use in academic and industrial laboratories.

Carrier polypeptide variants and/or target polypeptide variantscomprising one (or more) unnatural amino acid can be expressed andpurified from methylotrophic yeast. As described elsewhere herein,foreign genes can be expressed in P. pastoris from the alcohol oxidase 1(AOX1) promoter, the regulatory characteristics of which are well suitedfor this purpose. The AOX1 promoter is tightly repressed during growthof the yeast on most carbon sources, e.g., glycerol, glucose, orethanol, but is highly induced during growth on methanol (Tschorp et al.(1987) “Expression of the lacZ gene from two methanol-regulatedpromoters in Pichia pastoris.” Nucl Acids Res 15: 3859-3876). Expressionof, e.g., carrier polypeptide variants and/or target polypeptidevariants, encoded by genes regulated by P_(AOX1) can typically reach≧30% of the total soluble protein in P. pastoris cells grown onmethanol. For the production of recombinant carrier and/or targetpolypeptide variants, P_(AOX1)-controlled expression strains are growninitially on a repressing carbon source to generate biomass, e.g.,maximize culture density, and then shifted to a methanol-containingmedium, e.g., BMGY, BMMY, or BMM, as the sole energy source to induceexpression of the foreign gene.

However, promoters that are not induced by methanol can also beadvantageous for the expression of heterologous genes encoding carrierpolypeptides or polypeptide variants and/or target polypeptides orpolypeptide variants. Alternative promoters to the AOX1 promoter in thisexpression system are the P. pastoris GAP, FLD1, AOX2, ILC1, and YPT1promoters. Further details regarding the regulation of these promoters,the conditions under which it can be most beneficial to express aforeign gene from these promoters, and the expression of foreignproteins in P. pastoris by these promoters are discussed in, e.g., Searset al. (1998) “A Versatile Set of Vectors for Constitutive and RegulatedGene Expression in Pichia pastoris.” Yeast 14: 783-790; Vassileva et al.(2001) “Expression of hepatitis B surface antigen in the methylotrophicyeast Pichia pastoris using the GAP promoter.” J Biotechnology 88:21-35; Shen et al. (1998) “A strong nitrogen-source regulated promoterfor controlled expression of foreign genes in the yeast Pichiapastoris.” Gene 216: 93-102; Lin-Cereghino et al. “Expression of foreigngenes in the yeast Pichia pastoris.” Genetic Engineering Principles andMethods, Vol. 23 1^(st) Ed. Ed. Jane K. Setlow, Springer, N.Y.: (2005).

Although expression of carrier and/or target polypeptide variants in P.pastoris or other methylotrophic yeast can be done in shake-flaskculture, protein levels expressed in this system are typically muchhigher in fermenter cultures, because it is in fermenters thatparameters such as pH, aeration, and carbon source feed rate can becontrolled to achieve ultra-high cell densities, e.g., >100 g/L dry cellweight; >400 g/L wet cell weight, >500 OD₆₀₀ units/ml (see, e.g.,Lin-Cereghino et al. (2002) “Production of recombinant proteins infermenter cultures of the yeast Pichia pastoris.” Curr Opin Biotechnol13: 329-332. A hallmark of the P. pastoris expression system is the easewith which expression strains scale up from shake-flask to high-densityfermenter cultures.

A three step process can typically be employed to express carrier and/ortarget polypeptide variants encoded by genes under the transcriptionalcontrol of P-_(AOX1), in fermenter cultures of P. pastoris or othermethylotrophic yeast. In the first step, the engineered methylotrophicyeast expression strain is cultured in a simple, defined, mediumcomprising a non-fermentable, P_(AOX1)-repressing carbon source topermit the cell growth. The second step comprises a transition phaseduring which glycerol is fed to the culture at a growth-limiting rate tofurther increase the culture's biomass and to prepare the cells forinduction. During the third step, methanol is added to the culture at arate that allows the cells to physiologically acclimate to metabolizingmethanol and to synthesize the recombinant protein. The methanol feedrate is then adjusted upwards periodically until the desired growth rateand protein expression rate is achieved (Lin-Cereghino et al.“Expression of foreign genes in the yeast Pichia pastoris.” GeneticEngineering Principles and Methods, Vol. 23 1^(st) Ed. Ed. Jane K.Setlow, Springer, N.Y.: (2005)).

The media in which methylotrophic yeast can be grown are inexpensive andhighly defined, consisting of carbon sources, e.g., glycerol and/ormethanol, biotin, salts, trace elements, and water. The media are freeof pyrogens and toxins, and are therefore compatible with the productionof pharmaceutical agents for human use.

The recombinant carrier polypeptide variants and/or target polypeptidevariants expressed in methylotrophic yeast can be produced eitherintracellularly or extracellularly. Because methylotrophic yeast secreteonly low levels of endogenous protein, secreted recombinant protein canconstitute the majority of protein in the medium. Therefore, directingthe recombinant carrier and/or target polypeptide variant into theculture medium can serve as a first step in protein purification,eliminating the need to follow harsh yeast lysis protocols and avoidingthe possibility of contamination of the recombinant protein byendogenous yeast proteins. However, due to protein stability and foldingrequirements, secreting a heterologous protein into the medium istypically reserved only for those proteins that are normally secreted bytheir native host cells. Nevertheless, kits are available, e.g.,Original Pichia Expression Kit (Invitrogen), Multi-Copy PichiaExpression Kit (Invitrogen), Pichia Protein Expression System (ResearchCorporation Technologies), in which pre-made expression cassettes allowpractitioners to clone a gene of interest in frame with sequencesencoding its native secretion signal, the S. cerevisiae α-factor prepropeptide, or the P. pastoris acid phosphatase (PHO1) signal to allowsecretion into the culture medium. A number of techniques for therecovery of intracellular recombinant proteins from methylotrophic havealso been developed (Shepard et al. (2002) “Recovery of intracellularrecombinant proteins from the yeast Pichia pastoris by cellpermeabilization.” J Biotechnology 99: 149-160; U.S. Pat. No.6,821,752).

General Methods for the Purification of Heterologous Proteins fromMethylotrophic Yeast

A variety of protein purification methods are well known in the art andcan be applied to the purification and analysis of carrier and/or targetpolypeptides and polypeptide variants comprising UAA expressed inmethylotrophic yeast. These techniques, and others that are necessaryfor the analysis of polypeptides, include those set forth in R. Scopes,Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc. N.Y. (1990); Sandana (1997) Bioseparation of Proteins, AcademicPress, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker (1996) The Protein Protocols Handbook HumanaPress, NJ; Harris and Angal (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998) ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

Methods and Strategies for Strain Construction in Methylotrophic Yeast

Shuttle vectors that are suitable for replication in E. coli aretypically used to engineer nucleic acid constructs that place a gene ofinterest, e.g., a gene encoding a carrier polypeptide and/or a targetpolypeptide or variant thereof comprising a selector codon, under thecontrol of a highly inducible methylotrophic yeast promoter. Becauseplasmids are relatively unstable in methylotrophic yeast, the expressionconstructs are then usually linearized and transformed into e.g., aPichia cell, a Hansenula cell, a Candida cell, or a Torulopsis cell, andintegrated into the genome. Integration is generally site specific;however, high frequencies of non-homologous integration have beenobserved in Hansenula polymorpha (Agaphonov et al. (2005) “Defect ofvacuolar protein sorting stimulates proteolytic processing of humanurokinase-type plasminogen activator in the yeast Hansenula polymorpha.”FEMS Yeast Research 5: 1029-1035). Additional details regarding thegeneral molecular manipulation, e.g., transformation, gene targeting,cloning by functional complementation, use of available selectablemarkers, and the like, of methylotrophic yeast can be found in, e.g.;Peberdy, Ed. (1991) Applied Molecular Genetics of Fungi. CambridgeUniversity Press, UK; Hansenula Polymorpha: Biology and Applications,1st Ed., Wiley-VCH; Higgins and Cregg. Pichia Protocols (Methods inMolecular Biology), 1^(st) Ed. Humana Press: New Jersey (1998); and thereferences cited therein.

In a preferred embodiment, carrier polypeptide variants and/or targetpolypeptide variants, e.g., those described in detail elsewhere herein,are expressed in the methylotroph P. pastoris. Expression of mostforeign genes in P. pastoris can be performed by following three basicsteps: The insertion of the gene encoding the carrier or targetpolypeptide into an expression vector; the introduction of theexpression vector into the P. pastoris genome; and analysis of theputative expression strain for production of the carrier or targetpolypeptide variant expressed by the foreign gene, the methods for whichare described above. Fortunately, techniques for the molecular geneticmanipulation of P. pastoris, e.g., DNA-mediated transformation, genetargeting, gene replacement, and cloning by functional complementation,are similar to those described for S. cerevisiae. In contrast to S.cerevisiae, however, plasmids are unstable in P. pastoris, andexpression constructs encoding a protein of interest are insteadintegrated into the P. pastoris genome via homologous recombination.Protocols for the molecular genetic manipulation of P. pastoris arediscussed in detail in, e.g., Cregg et al. (1985) “Pichia pastoris as ahost system for transformations.” Molec Cell Biol 5: 3376-3385;Lin-Cereghino et al. “Expression of foreign genes in the yeast Pichiapastoris.” Genetic Engineering Principles and Methods, Vol. 23 1^(st)Ed. Ed. Jane K. Setlow, Springer, NY: (2005); Higgins and Cregg. PichiaProtocols (Methods in Molecular Biology), 1^(st) Ed. Humana Press: NewJersey (1998); Lin-Cereghino et al. (2000) “Heterologous proteinexpression in the methylotrophic yeast Pichia pastoris.” FEMS MicrobiolRev 24: 45-66, and in the references cited therein.

A variety of P. pastoris host strains and expression vectors areavailable. Virtually all P. pastoris expression strains are derived fromNRRL-Y 111430 (Northern Regional Research Laboratories, Peoria, Ill.).Most expression strains have one or more auxotrophic markers that permitselection of expression vectors comprising the appropriate complementarymarkers. Host strains can differ in their abilities to metabolizemethanol because of deletions in AOX1, AOX2, or both. In fact, strainscarrying mutations in AOX1 and/or AOX2 can be better producers offoreign proteins than wild-type strains (Cregg et al. (1987) “High levelexpression and efficient assembly of hepatitis B antigen in themethylotrophic yeast, Pichia pastoris.” Bio/Technology 5: 479-485;Chiruvolu et al. (1997) “Recombinant protein production in an alcoholoxidase-defective strain of Pichia pastoris in fed-batch fermentation.”Enzyme Microb Technol 21: 277-283). Nevertheless, even aox1⁻ strainsretain the ability to induce expression of foreign proteins at highlevels from the AOX1 promoter. More detailed information host strains,including protease deficient host strains in which the expression ofcertain recombinant proteins may be more beneficial, is available in,e.g., Brierley et al. (1998) “Secretion of recombinant insulin-likegrowth factor-1 (IGF-1).” Methods Mol Biol 103: 149-177; White et al.(1995) “Large-scale expression, purification, and characterization ofsmall fragments of thrombomodulin: the roles of the sixth domain and ofmethionine 388.” Protein Eng 8: 1177-1187.

Most P. pastoris expression vectors have been designed as E. coli/P.pastoris shuttle vectors, containing origins of replication formaintenance in E. coli and selectable markers that are functional in oneor both organisms, e.g., ARG4, HIS4, ADE1, URA3, TRP1 and certainantibiotics, e.g., Zeocin™ and Geneticin®, which are selectable in P.pastoris, and/or any of a number of antibiotic resistance markers whichare selectable in E. coli. Typically, an expression vector will comprise5′ AOX1 promoter sequences and AOX1-derived sequences fortranscriptional termination, between which lies a multiple cloning site.Although the AOX1 promoter has been successfully used to expressnumerous foreign proteins, there are circumstances under which the useof this promoter may not be suitable, e.g., for the production of foodproducts. Alternative promoters to the AOX1 promoter in this expressionsystem are the P. pastoris AOX2, ICL1, GAP, FLD1, and YPT1 promoters.Generalized diagrams of expression vectors comprising any of theaforementioned promoters and lists of possible vector components arealso given in, e.g., Lin-Cereghino et al. “Expression of foreign genesin the yeast Pichia pastoris.” Genetic Engineering Principles andMethods, Vol. 23 1^(st) Ed. Ed. Jane K. Setlow, Springer, N.Y.: (2005)and Lin-Cereghino, et al. (2000) “Heterologous protein expression in themethylotrophic yeast Pichia pastoris.” FEMS Microbiol Rev 24: 45-66. Inaddition, the DNA sequences of many vectors can be found at theInvitrogen website (www.invitrogen.com), and are available fromInvitrogen individually and in P. pastoris expression kits.

General Molecular Cloning Methods and Techniques

Procedures for isolating, cloning, and amplifying nucleic acids inpreparation for, e.g., cloning a gene of interest, e.g., a gene encodinga carrier polypeptide variant or a target polypeptide variant, into anexpression construct as described above, are replete in the literatureand can be used in the present invention to, e.g., provide and express agene of interest in a methylotrophic yeast, e.g., P. pastoris. Furtherdetails these techniques can be found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. MolecularCloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); The NucleicAcid Protocol's Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor,Humana Press Inc (Rapley); Current Protocols in Molecular Biology, F. M.Ausubel et al. eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2007) (“Ausubel”)); PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis); Chen et al. (ed) PCR Cloning Protocols, Second Edition(Methods in Molecular Biology, volume 192) Humana Press; in Viljoen etal. (2005) Molecular Diagnostic PCR Handbook Springer; and Demidov andBroude (eds) (2005) DNA Amplification: Current Technologies andApplications. Horizon Bioscience, Wymondham, UK. Other usefulreferences, e.g., for cell isolation and culture, e.g., for subsequentnucleic acid isolation, include Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

A plethora of kits are also commercially available for the purificationof plasmids or other relevant nucleic acids from cells, (see, e.g.,EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™, fromStratagene; QIAprep™ from Qiagen). Any isolated and/or purified nucleicacid can be further manipulated to produce other nucleic acids, used totransfect cells, incorporated into related vectors to infect organismsfor expression, and/or the like. Typical cloning vectors containtranscription and translation terminators, transcription and translationinitiation sequences, and promoters useful for regulation of theexpression of the particular target nucleic acid. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems. SeeSambrook, Ausubel and Berger. In addition, essentially any nucleic acidcan be custom or standard ordered from any of a variety of commercialsources, such as Operon Technologies Inc. (Huntsville, Ala.).

It will be appreciated that while particular methods of constructingcarrier and/or target polypeptide variants that comprise chemicallyreactive unnatural amino acids are detailed herein, e.g., usingorthogonal translation systems in methylotrophic yeast, they should notnecessarily be taken as limiting. Carrier polypeptide variantscomprising a first reactive amino acid and/or target polypeptidevariants comprising a second reactive amino acid can be constructedusing orthogonal translation systems in, e.g., E. coli, S. cerevisiae,mammalian cells, etc. Furthermore, other, e.g., non-orthogonal, methodsof constructing carrier and/or target polypeptides having unnaturalamino acids are also included herein in the many embodiments. Suchmethods are described in further detail below.

Non-Orthogonal Methods for the Direct Incorporation of Unnatural AminoAcids into Carrier Polypeptides and/or Target Polypeptides

As stated above, in different embodiments of the invention, carrierpolypeptides and target polypeptides or carrier polypeptides and targetpolypeptides variants (that each comprise an reactive unnatural aminoacid which, when reacted with the unnatural amino acid in the other,forms a stable carrier polypeptide-target polypeptide conjugate) can beconstructed via direct incorporation methods such as an orthogonaltranslation system. This represents a preferred embodiment, due to theability of orthogonal systems to produce high yields of correctly foldedand post-translationally modified polypeptides with site-specificallyincorporated unnatural amino acids. Alternatively or additionally,however, other strategies for the direct incorporation of unnaturalamino acids into a polypeptide chain can be employed to introduce firstand second unnatural amino acids into the carrier polypeptide variantsand/or target polypeptide variants, respectively. It will be appreciatedthat in typical embodiments herein, an unnatural amino acid isincorporated into a carrier and/or target polypeptide duringconstruction of the polypeptide and is not added via post-translationalchemical derivatization.

For example, one general in vitro biosynthetic method for incorporatingunnatural amino acids into, e.g., carrier and/or target polypeptides,during primary construction uses nonsense or frameshift suppressor tRNAsthat have been chemically acylated with the desired unnatural amino acidand then added to an in extract capable of supporting proteinbiosynthesis and which includes a gene containing a desired ambernonsense mutation. This strategy has been used to site-specificallyincorporate over 100 unnatural amino acids into a variety of proteins ofvirtually any size and can be used herein to create carrier and/ortarget polypeptide variants that comprise unnatural amino acids. See,e.g., Cornish et al. (1995) “Probing Protein Structure and Function withan Expanded Genetic Code.” Angew Chem Int Ed Engl 34: 621-633; Noren etal. (1989) “A general method for site-specific incorporation ofunnatural amino acids into proteins.” Science 244: 182-188; and Bain etal. (1989) “Biosynthetic site-specific incorporation of a non-naturalamino acid into a polypeptide.” JACS 111: 8013-8014.

In other embodiments, unnatural amino acids can be directly incorporatedinto smaller carrier and/or target polypeptides (ranging from 60-100amino acids) via chemical synthesis. Solid phase peptide synthesis is amethod that is widely used to chemically synthesize peptides and smallproteins that comprise unnatural amino acids (see, e.g., Merrifield(1963) “Solid Phase Peptide synthesis. I. The synthesis of atetrapeptide.” JACS 85: 2149-2154) and can be adapted to produce carrierand/or target polypeptides comprising unnatural amino acids that can bereacted to produce a stable conjugate. This technique typicallycomprises two stages: The first stage solid phase peptide synthesis(SPPS) includes the assembly of a peptide chain using protected aminoacid derivatives on a polymeric support via repeated cycles ofcoupling-deprotection. The free N-terminal amine of a solid-phaseattached peptide can then be coupled to a single N-protected amino acidunit, e.g., an unnatural amino acid. This unit is then deprotected,revealing a new N-terminal amine to which a further amino acid may beattached. While the peptide is being synthesized usually by stepwisemethods, all soluble reagents can be removed from the peptide-solidsupport matrix by filtration and washed away at the end of each couplingstep. In the second stage of SPPS, the peptide is cleaved from thesupport and side-chain protecting groups are removed to produce thepeptide, e.g., a carrier or target polypeptide comprising one or moreunnatural amino acids. There are two major used forms of solid phasepeptide synthesis: Fmoc (Carpino et al. (1972)“9-Fluorenylmethoxycarbonyl amino-protecting group.” J Org Chem 37:3404-3409), in which a base labile alpha-amino protecting group is used,and t-Boc, in which an acid labile protecting group is used. Each methodinvolves different resins and amino acid side-chain protection andconsequent cleavage/deprotection steps. For additional details regardingpeptide synthesis, see the following publications and references citedwithin: Crick et al. (1961) “General nature of the genetic code forproteins.” Nature 192: 1227-1232; Hofmann et al. (1966) “Studies onPolypeptides. XXXVI. The Effect of Pyrazole-Imidazole Replacements onthe S-Protein Activating Potency of an S-Peptide Fragment¹⁻³ .” JACS 88:5914-5919; Kaiser et al. (1989) “Synthetic approaches to biologicallyactive peptides and proteins including enzymes.” Acc Chem Res 22: 47-54;Nakatsuka et al. (1987) “Peptide segment synthesis catalyzed by thesemisynthetic enzyme thiolsubtilisin.” JACS 109: 3808-3810; Schnolzer etal. (1992) “Constructing proteins by dovetailing unprotected syntheticpeptides: backbone-engineered HIV protease.” Science 5054: 221-225;Chaiken et al. (1981) “Semisynthetic peptides and proteins.” CRC Crit.Rev Biochem 11: 255-301; Offord (1987) “Protein engineering by chemicalmeans?” Protein Eng 1: 151-157; and Jackson et al. “A designed peptideligase for total synthesis of ribonuclease A with unnatural catalyticresidues.” Science 5184: 243-247.

Chemical Coupling Reactions that can be Used to Conjugate CarrierPolypeptide and Target Polypeptide Variants

Current methods for chemically coupling target polypeptide variants(e.g., small therapeutic peptide variants) to carrier polypeptidevariants range from the use of non-specific reagents, e.g.,glutaraldehyde or carbodiimide activated N-hydroxysuccinimide esters, tohighly specific heterobifunctional crosslinkers that can circumvent theformation of carrier polypeptide-carrier polypeptide ortarget-polypeptide-target polypeptide conjugates. However, only alimited number of amino acids can be chemically modified with suchreagents (e.g., amino acids comprising amine, keto, thiol, sulfhydryl,or carboxyl groups). Using these reagents in the coupling of a carrierpolypeptide to a target polypeptide can perturb the conformation of thecarrier polypeptide, the target polypeptide, or the resultingcarrier-target polypeptide conjugate, thus decreasing the conjugate'sstability, biological activity, pharmacokinetic activity, etc. Usingsuch reagents can often result in the production of a heterogeneouspopulation of carrier-polypeptide-target polypeptide complexes, whichcan be difficult to separate, decreasing manufacturing efficiency andcomplicating quality control.

In contrast, conjugates of the invention are produced by reacting afirst unnatural amino acid that has been incorporated into a carrierpolypeptide variant, e.g., using an orthogonal translation system in amethylotrophic yeast cell, with a second unnatural amino acid that hasbeen incorporated into a target polypeptide variant in an orthogonalcoupling reaction, e.g., any one of the chemical ligation reactionsdescribed below. These reactions can be performed in vitro or in vivo,depending on the appropriate reaction conditions. Because only theunnatural amino acids present on the carrier and target polypeptidevariants participate in the ligation reaction, the methods of theinvention can be reliably used to produce homogenous populations ofwell-defined carrier-polypeptide-target polypeptide conjugates (e.g.,comprising defined stoichiometries and defined ligation sites) with highefficiency. Because any of a variety chemically of reactive first andsecond unnatural amino acids can optionally be incorporated into carrierand target polypeptide variants, respectively, the production ofcarrier-target polypeptide conjugates is not limited only to thoseconditions under which a particular chemical ligation reaction canproceed, e.g., conditions under which the target polypeptide variant,the carrier polypeptide variant, or the resulting carrier-targetconjugate may be unstable. Furthermore, existing technologiesbeneficially permit the incorporation of unnatural amino acids into anyamino acid position in a polypeptide. Thus, placement of the first andsecond chemically reactive unnatural amino acids in the carrier andtarget polypeptides, respectively, can optionally be chosen based on,e.g., whether placement in that location would change, e.g., theconformations, biological activities, pharmacological activities,stabilities, bioavailabilities, or other properties, of the carrierpolypeptide, of the target polypeptide, or of the resultingcarrier-target polypeptide conjugate.

In one illustrative, but non-limiting example described in more detailbelow, a p-acetylphenylalanine, which has been incorporated into HSA,can be reacted with a ε-(2-(aminooxy)acetyl)-L-lysine, which has beenincorporated into ABT-510, via oxime ligation to produce an HSA-ABT-510conjugate with an increased serum half-life. It will be appreciated thatillustrations in the Example below are not the only embodiments of theinvention. As will be apparent from the description herein, any of awide variety of chemical ligation reactions can be used to couple acarrier polypeptide that includes a first reactive unnatural amino acidto a target polypeptide that includes a second reactive unnatural aminoacid, wherein the coupling entails reacting the first and secondunnatural amino acids.

In various embodiments of the invention, first and second unnaturalamino acid are optionally reacted via one or more of anelectrophile-nucleophile reaction, an oxime ligation, a ketone reactionwith a nucleophile, an aldehyde reaction with a nucleophile, a reactionbetween a carbonyl group and a nucleophile, a reaction between asulfonyl group and a nucleophile, an esterification reaction, a reactionbetween a hindered ester group and a nucleophile, a reaction between athioester group and a nucleophile, a reaction between a stable iminegroup and a nucleophile, a reaction between an epoxide group and anucleophile, a reaction between an aziridine group and a nucleophile, areaction between an electrophile and an aliphatic or aromatic amine, areaction between an electrophile and a hydrazide, a reaction between anelectrophile and a carbohydrazide, a reaction between an electrophileand a semicarbazide, a reaction between an electrophile and athiosemicarbazide, a reaction between an electrophile and acarbonylhydrazide, a reaction between an electrophile and athiocarbonylhydrazide, a reaction between an electrophile and asulfonylhydrazide, a reaction between an electrophile and a carbazide, areaction between an electrophile and a thiocarbazide, a reaction betweenan electrophile and a hydroxylamine, a reaction between a nucleophile ornucleophiles such as a hydroxyl or diol and a boronic acid or ester, atransition metal catalyzed reaction, a palladium catalyzed reaction, acopper catalyzed heteroatom alkylation reaction, a cycloadditionreaction, a 1,3, cycloaddition reaction, a 2,3 cycloaddition reaction,an alkyne-azide reaction, a Diels-Alder reaction, or a Suzuki couplingreaction. Some of these reactions are described in further detail below.

Oxime Ligation

Oxime ligation was first used in the chemoselective ligation ofunprotected polypeptides in an effort to produce a syntheticmacromolecule of controlled structure of a molecular weight greater than10kD (Rose (1994) “Facile Synthesis of Homogenous Artificial Proteins.”JAGS 116: 30-33). In a first step, a purified polypeptide that carriesan aldehyde group and a second purified polypeptide that carries anaminooxy group are prepared. In a second step, the two polypeptidesspontaneously self-assemble under very mild conditions through formationof an oxime bond. The resulting oximes are stable in water at roomtemperature at pH 2-7.

As described in the Example below, an HSA-ABT-510 conjugate was producedby reacting the aminooxy group of a ε-(2-(aminooxy)acetyl)-L-lysineresidue in an ABT-510 variant with the keto group of ap-acetylphenylalanine residue in an HSA variant at a pH<5. The aminooxygroup undergoes a selective oxime ligation with the keto group tocovalently link the ε-(2-(aminooxy)acetyl)-L-lysine to thep-acetylphenylalanine, thus covalently coupling the HSA to the ABT-510.

1,3-Dipolar Cycloaddition Reactions

1,3-dipolar cycloaddition, also known as “click” chemistry, is thereaction between a 1,3-dipole and a dipolarophile, e.g., a substitutedalkene, to form a five-membered ring. One useful example of a 1,3dipolar cycloadditon is the Azide-Alkyne Huisgen Cycloaddition, e.g., a1,3-dipolar cycloaddition between an azide and a terminal or internalalkyne to give a 1,2,3-triazole. This copper(I)-catalyzed reaction ismild and very efficient, requiring no protecting groups, and requiringno purification in many cases (Rostovtsev et al. (2002) “A StepwiseHuisgen Cycloaddition Process: Copper(I)-Catalyzed RegioselectiveLigation of Azides and Terminal Alkynes” Angew Chem Int Ed 41:2596-2599). Because this reaction involves a cycloaddition rather than anucleophilic substitution, proteins can be modified with extremely highselectivity. This reaction can be carried out at room temperature inaqueous conditions with excellent regioselectivity (1,4>1,5) by theaddition of catalytic amounts of Cu(I) salts to the reaction mixture.See, e.g., Tornoe et al. (2002) “Peptidotriazoles on solid phase:[1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolarcycloadditions of terminal alkynes to azides.” J Org Chem 67: 3057-3064;and Rostovtsev et al. (2002) “A Stepwise Huisgen Cycloaddition Process:Copper(I)-Catalyzed Regioselective Ligation of Azides and TerminalAlkynes” Angew Chem Int Ed 41: 2596-2599. The azide and alkynefunctional groups are largely inert towards biological molecules andaqueous environments, which allows the use of the Azide-Alkyne HuisgenCycloaddition in the coupling of, e.g., carrier polypeptides to targetpolypeptides. The triazole has similarities to the ubiquitous amidemoiety found in nature, but unlike amides, is not susceptible tocleavage. Additionally, triazoles are nearly impossible to oxidize orreduce.

Suzuki Coupling Reactions

A target polypeptide variant (or carrier polypeptide variant) comprisingan surface-exposed unnatural amino acid with an aryl iodide can becovalently attached to a carrier polypeptide variant (or targetpolypeptide variant) comprising a surface-exposed boronic unnaturalamino acid residue, e.g., a p-boronophenylalanine, anm-boronophenylalanine, or an o-boronophenylalanine, through a palladiumcatalyzed Suzuki coupling. For a description of this chemistry, see,e.g., Miyaura and Suzuki (1995) “Palladium-Catalyzed Cross-CouplingReactions of Organoboron Compounds,” Chemical Reviews 95: 2457 andSuzuki (1999) “Recent advances in the cross-coupling reactions oforganoboron derivatives with organic electrophiles, 1995-1998,” Journalof Organometallic Chemistry 576:147. Aryl iodides are reactive groupswith a variety of uses in organometallic chemistry, includingsilylation, aminocarbonylation, Heck Arylation, vinylation,cross-coupling with aryl acetylenes, and many others. Further detailsregarding the use of unnatural amino acids in Suzuki coupling reactionsare elaborated in U.S. patent application Ser. No. 12/262,025, entitled,“A Genetically Encoded Boronate Amino Acid”, filed Oct. 30, 2008 andInt'l Application PCT/US2008/081868 entitled “A Genetically EncodedBoronate Amino Acid,” filed Oct. 30, 2008.

Copper-Catalyzed Heteroatom Alkylation Reactions

A target polypeptide variant or carrier polypeptide variant thatcomprises an unnatural amino acid residue that includes a boronate groupcan participate in copper catalyzed heteroatom alkylation reactions(Chan et al. (2003) “Copper promoted C—N and C—O bond cross-couplingwith phenyl and pyridylboronates.” Tetrahedron Letters 44: 3863),asymmetric reductions (Huang et al (2000) “Asymmetric reduction ofacetophenone with borane catalyzed by chiral oxazaborolidinone derivedfrom L-α-amino acids.” Synthetic Communications 30: 2423), Diels-Alderreactions (Ishihara and Yamamoto (1999) “Arylboron Compounds as AcidCatalysts in Organic Synthetic Transformations.” European Journal ofOrganic Chemistry 3: 527-538), as well as a variety of othertransformations.

A boronic unnatural amino acid residue present on the surface of acarrier or target polypeptide variant can also be used to formreversible boronic esters with unnatural amino acid residues on acorresponding target or carrier polypeptide variant that includes analcohol group a diol an amino-alcohol, or a diamine containing moiety.For example, boronic acids form reversible covalent complexes withdiols. For an early description of this chemistry, see Lorand andEdwards (1959) “Polyol Complexes and Structure of the BenzeneboronateIon.” Journal of Organic Chemistry 24: 769-774. Reversible complexes canalso be formed with aminoalcohols (Springsteen et al. (2001) “TheDevelopment of Photometric Sensors for Boronic Acids.” BioorganicChemistry 29: 259-270), amino acids (Mohler and Czarnik (1994)“Alpha-Amino Acid Chelative Complexation by an Arylboronic Acid.[Erratum to document cited in CA119(17):181171a].” JACS 116: 2233;Mohler and Czarnik (1993) “Alpha-Amino-Acid Chelative Complexation by anArylboronic Acid,” JACS 115: 7037-7038) alkoxides (Cammidge and Crépy(2004) “Synthesis of chiral binaphthalenes using the asymmetric Suzukireaction.” Tetrahedron 60: 4377-4386), and hydroxamic acids (Lamandé etal. (1980) “Structure et acidite de composes a atome de bore et dephosphore hypercoordonnes,” Journal of Organometallic Chemistry 329:1-29. Further details regarding the utility of boronic amino acids inchemical ligation reactions can be found in U.S. patent application Ser.No. 12/262,025, entitled, “A Genetically Encoded Boronate Amino Acid”,filed Oct. 30, 2008; Int'l Application PCT/US2008/081868 entitled “AGenetically Encoded Boronate Amino Acid,” filed Oct. 30, 2008; andreferences cited therein.

[2+3] Cycloaddition Reactions

In certain embodiments, a carrier polypeptide variant comprising a firstunnatural amino acid can be coupled to target polypeptide variantcomprising a second unnatural amino acid through a [2+3] cycloaddition.In one embodiment, the first unnatural amino acid includes an alkynyl orazido moiety and the second unnatural amino acid includes an azido oralkynyl moiety. For example, the first unnatural amino acid includes thealkynyl moiety (e.g., in unnatural amino acidp-propargyloxyphenylalanine) and the second unnatural amino acidincludes the azido moiety. In another example, the first unnatural aminoacid includes the azido moiety (e.g., in the unnatural amino acidp-azido-L-phenylalanine) and the second unnatural amino acid includesthe alkynyl moiety. The use of unnatural amino acids in [2+3]cycloaddition reactions is described in U.S. patent application Ser. No.10/826,919, entitled, “Unnatural Reactive Amino Acid Genetic CodeAdditions”, filed Apr. 4, 2004.

Electrophile-Nucleophile Reactions

In some embodiments, one of the reactive groups present in an unnaturalamino acid that has been incorporated into a carrier polypeptide variant(or target polypeptide variant) is an electrophilic moiety, and thereactive group present in a second unnatural amino acid that has beenincorporated into a target polypeptide variant (or carrier polypeptidevariant) is a nucleophilic moiety. Suitable electrophilic moieties thatreact with nucleophilic moieties to form a covalent bond are known tothose of skill in the art. Such electrophilic moieties include, but arenot limited to, e.g., carbonyl group, a sulfonyl group, an aldehydegroup, a ketone group, a hindered ester group, a thioester group, astable imine group, an epoxide group, an aziridine group, etc.

The product of the reaction between the nucleophile and the electrophiletypically incorporates the atoms originally present, e.g., in thenucleophilic moiety. In some embodiments, the electrophile is analdehyde or ketone with the nucleophilic moiety including reactionproducts such as an oxime, an amide, a hydrazone, a reduced hydrazone, acarbohydrazone, a thiocarbohydrazone, a sulfonylhydrazone, asemicarbazone, a thiosemicarbazone, or similar functionality, dependingon the nucleophilic moiety used and the electrophilic moiety (e.g.,aldehyde, ketone, and/or the like) that is reacted with the nucleophilicmoiety. Linkages with carboxylic acids are typically referred to ascarbohydrazides or as hydroxamic acids. Linkages with sulfonic acids aretypically referred to as sulfonylhydrazides or N-sulfonylhydroxylamines.The resulting linkage can be subsequently stabilized by chemicalreduction.

Suitable nucleophilic moieties that can react with aldehydes and ketonesto form a covalent bond are known to those of skill in the art. Suchnucleophiles include, for example, aliphatic or aromatic amines, such asethylenediamine. In other embodiments, the unnatural amino acid caninclude reactive groups such as —NR¹—NH₂ (hydrazide), —NR¹(C═O)NR²NH₂(semicarbazide), —NR¹(C═S)NR²NH₂ (thiosemicarbazide), —(C═O)NR¹NH₂(carbonylhydrazide), —(C═S)NR¹NH₂ (thiocarbonylhydrazide), —(SO₂)NR¹NH₂(sulfonylhydrazide), —NR¹NR²(C═O)NR³NH₂ (carbazide), —NR¹NR²(C═S)NR³NH₂(thiocarbazide), or —O—NH₂ (hydroxylamine), where each R¹, R², and R³ isindependently H, or alkyl having 1-6 carbons, preferably H. In oneaspect of the invention, the reactive group is a hydrazide,hydroxylamine, carbohydrazide or a sulfonylhydrazide.

Still other reactive chemistries also find use with the invention,including but not limited to the Staudinger ligation and the olefinmetathesis chemistries (see, e.g., Mahal et al. (1997) “EngineeringChemical Reactivity on Cell Surfaces Through OligosaccharideBiosynthesis.” Science 276: 1125-1128). Many other coupling chemistriesare also applicable, and can be used, depending on the unnatural aminoacids to be incorporated into the carrier and target polypeptide. Suchreactions are well known to those of skill in the are and are describedin further detail in, e.g., Dawson et al. (1994) “Synthesis of Proteinsby Native Chemical Ligation.” Science 266: 776-779; Lemieux et al.(1998) “Chemoselective ligation reactions with proteins,oligosaccharides and cells.” TIBS 16: 506-513; Knipe, Chris. OrganicReaction Mechanisms, 2004. New York: Wiley, 2004; and others.

Pharmaceutical Compositions and their Administration

The carrier polypeptide-target polypeptide conjugates of the inventionare optionally employed for therapeutic uses, e.g., in combination witha suitable pharmaceutical carrier. Such compositions comprise, e.g., atherapeutically effective amount of the conjugate, and apharmaceutically acceptable carrier or excipient. Such a carrier orexcipient includes, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and/or combinations thereof. Theformulation is made to suit the mode of administration. In general,methods of administering proteins are well known in the art and can beapplied to administration of the conjugates of the invention.

Therapeutic compositions comprising one or more carrierpolypeptide-target polypeptide conjugates of the invention areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, to confirm efficacy, tissue metabolism, and toestimate dosages, according to methods well known in the art. Inparticular, dosages can be initially determined by activity, stabilityor other suitable measures of unnatural herein to unconjugated targetproteins (e.g., comparison of a carrier polypeptide-target peptideconjugate, e.g., an HSA-TSP-1 conjugate (or an HSA-ABT-510 conjugate),to a TSP (or ABT-510) that is not conjugated to an HSA and which doesnot comprise any unnatural amino acids), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. Thecarrier-peptide/target polypeptide conjugates of the invention areadministered in any suitable manner, optionally with one or morepharmaceutically acceptable carriers. Suitable methods of administeringsuch conjugates in the context of the present invention to a patient areavailable, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. Pharmaceutically acceptable carriers and excipientsare well known in the art, and one or more conjugates of the inventioncan be formulated into pharmaceutical compositions by well-known methods(see, e.g., Remington: The Science and Practice of Pharmacy, 21stedition, A. R. Gennaro, Ed., Mack Publishing Company (2005);Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000); and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress (2000)).

Carrier polypeptide-target polypeptide conjugates of the invention canbe administered by a number of routes including, but not limited to:oral, intravenous, intraperitoneal, intramuscular, transdermal,subcutaneous, topical, sublingual, or rectal means. Carrierpolypeptide-target polypeptide conjugates can also be administered vialiposomes. Such administration routes and appropriate formulations aregenerally known to those of skill in the art.

The conjugates of the invention, alone or in combination with othersuitable components, can also be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for carrier polypeptide-target polypeptide conjugatetherapeutics, along with formulations in current use, provide preferredroutes of administration and formulation for the conjugates of theinvention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to effect a beneficial therapeutic response inthe patient over time, or, e.g., to inhibit infection by a pathogen, toreduce or prevent the symptoms of a disease state, or other appropriateactivity, depending on the application. The dose is determined by theefficacy of a particular composition/formulation, and the activity,stability or serum half-life of the carrier polypeptide-targetpolypeptide conjugate employed and the condition of the patient, as wellas the body weight or surface area of the patient to be treated. Thesize of the dose is also determined by the existence, nature, and extentof any adverse side-effects that accompany the administration of aparticular composition/formulation, or the like in a particular patient.

In determining the effective amount of the composition/formulation to beadministered in the treatment or prophylaxis of disease (e.g., cancers,inherited diseases, diabetes, AIDS, or the like), the physicianevaluates circulating plasma levels, formulation toxicities, progressionof the disease, and/or where relevant, the production of anti-unnaturalamino acid polypeptide antibodies.

The dose administered, e.g., to a 70 kilogram patient, is typically inthe range equivalent to dosages of currently-used therapeutic proteins,adjusted for the altered activity or serum half-life of the relevantcarrier polypeptide-target polypeptide conjugate. Thecompositions/formulations of this invention can supplement treatmentconditions by any known conventional therapy, including antibodyadministration, vaccine administration, administration of cytotoxicagents, natural amino acid polypeptides, nucleic acids, nucleotideanalogues, biologic response modifiers, and the like.

For administration, formulations of the conjugates of the presentinvention are administered at a rate determined by the LD-50 of therelevant formulation, and/or observation of any side-effects of thecarrier polypeptide-target polypeptide conjugate at variousconcentrations, e.g., as applied to the mass and overall health of thepatient. Administration can be accomplished via single or divided doses.General Methods for preparing administrable compositions are known tothose skilled in the art and are described in more detail in e.g.,Remington: The Science and Practice of Pharmacy, 21st edition, A. R.Gennaro, Ed., Mack Publishing Company (2005).

If a patient undergoing infusion of a formulation comprising one or moreconjugates of the invention develops fevers, chills, or muscle aches,he/she receives the appropriate dose of aspirin, ibuprofen,acetaminophen or other pain/fever controlling drug. Patients whoexperience reactions to the infusion such as fever, muscle aches, andchills are premedicated 30 minutes prior to the future infusions witheither aspirin, acetaminophen, or, e.g., diphenhydramine. Meperidine isused for more severe chills and muscle aches that do not quickly respondto antipyretics and antihistamines. Treatment is slowed or discontinueddepending upon the severity of the reaction.

A variety of subjects can benefit from the therapeutic treatments,and/or prophylactic treatments provided by the carrierpolypeptide-target polypeptide conjugates provided by the invention.Humans, and such animals including, but not limited to, domesticlivestock, such as cows, pigs, goats, sheep, chickens, and/or othercommon farm animals can be administered compositions and formulationsthat include the conjugates described herein. Common household pets,e.g., cats, dogs, parrots, parakeets, etc., can also benefit from beingadministered a therapeutic or prophylactic carrier polypeptide-targetpolypeptide conjugate.

Further details regarding the use of animal models and animal subjects,in biomedical testing and veterinary treatment are elaborated in, e.g.,Ng, Chow, and Ogden, eds. Using Animal Models in Biomedical Research: APrimer for the Investigator. First Edition. Singapore: World ScientificPublishing Company, 2008; Conn, ed. Sourcebook of Models for BiomedicalResearch. Totowa, N.J.: Springer, 2008; Woodhead, ed. NonmammalianAnimal Models for Biomedical Research (Vol 1). New York: Academic Press,1990. See also, e.g., Adams, ed. Veterinary Pharmacology andTherapeutics. Eighth Edition. USA: Wiley-Blackwell, 2001; Kahn and Line,Eds. Merck Veterinary Manual. Ninth Edition. USA: Merck, 2005; andreferences cited therein.

Carrier polypeptide-target polypeptide conjugates provided by theinvention can be administered not only to treat a disease state in asubject, e.g., a human, but also to perform treatment efficacy tests, aswell as metabolic tests, toxicology tests, and specific tests todetermine the effects of the carrier peptide-target polypeptideconjugates on reproductive function or embryonic toxicity, or todetermine their carcinogenic potential. Performing such observationalstudies can entail administering the conjugates of the invention to avariety of animal subjects. Those of skill in the art will be quitefamiliar with numerous medical tests and measurements to help inselection of animal subjects that are to be administered thecompositions/formulations that include the conjugates of the invention.Such animal subjects include, but are not limited to, e.g., mammals suchas goats sheep, camels, cows, pigs, rabbits, horses, hamsters, non-humanprimates (monkeys, including cynomolgus monkeys, baboons, Old WorldMonkeys, and chimpanzees), guinea pigs, rats, mice, and/or cats. Birdssuch as, e.g., domestic fowl (chickens, turkeys), cockatiels, psittacinebirds, and cage and/or aviary birds, as well as bird embryos, can alsobe used in the research and development, production, quality control, orsafety testing of the carrier polypeptide-target polypeptide conjugatesprovided by the invention.

Fish, such as zebrafish, platyfish, and swordtails; amphibians,including, e.g., frogs and salamanders; and reptiles (snakes, lizards,and turtles) can also be used in a wide variety of tests to determinethe safety, effective dose, and/or toxicology of the compositionsdescribed herein and/or the methods of their administration. See, e.g.,Barry, et al. (2002) “Information Resources for Reptiles, Amphibians,Fish, and Cephalopods Used in Biomedical Research.” United StatesDepartment of Agriculture National Agricultural Library Animal WelfareInformation Center, and the references cited therein.

Kits and Articles of Manufacture

Kits are also a feature of the invention. For example, kits canoptionally contain any one or more carrier polypeptide-targetpolypeptide conjugates provided by the invention. Alternatively oradditionally, kits can contain reagents for the synthesis of carrierpolypeptide variants that comprise first chemically reactive unnaturalamino acids and/or target polypeptides variants, e.g., small therapeuticpeptides, that comprise second chemically reactive unnatural aminoacids. Such reagents can include, e.g., the reactive unnatural aminoacids, host cells, e.g., methylotrophic yeast cells that includeorthogonal translation system components suitable for the production ofcarrier polypeptide and/or target polypeptide variants comprisingunnatural amino acids, solutions in which to perform ligation reactionsthat produce the conjugates of the invention, reagents with which toproduce therapeutic formulations comprising one or more conjugates ofthe invention, media, etc. Kits of the invention can include additionalcomponents such as instructions to, e.g., construct a methylotrophicyeast strain that can express a carrier polypeptide and/or targetpolypeptide that comprises unnatural amino acids, perform a chemicalligation reaction to produce a carrier polypeptide-target polypeptideconjugate, etc. The kit can include a container to hold the kitcomponents, instructional materials for practicing any method or anycombination of methods herein, instructions for using cells (e.g.,methylotrophic yeast cells) provided with the kit, e.g., to produce acarrier and/or target polypeptide of interest that comprises achemically reactive unnatural amino acid at a selected amino acidposition.

EXAMPLE

The following example is offered to illustrate, but not to limit theclaimed invention. It is understood that the embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

Expanding the Genetic Repertoire of the Methylotrophic Yeast, Pichiapastoris

To increase the utility of protein mutagenesis with unnatural aminoacids, a recombinant expression system in the methylotrophic yeastPichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA(aaRS/tRNA_(CUA)) pairs previously evolved in Saccharomyces cerevisiaeto be specific for unnatural amino acids were inserted betweeneukaryotic transcriptional control elements and stably incorporated intothe P. pastoris genome. Both the Escherichia coli tyrosyl- andleucyl-RS/tRNA_(CUA) pairs were shown to be orthogonal in P. pastorisand used to incorporate eight unnatural amino acids in response to anamber codon with high yields and fidelities. One example shows that arecombinant human serum albumin mutant containing a keto amino acid(p-acetylphenylalanine) (FIG. 6 b, structure 1) can be efficientlyexpressed in this system and selectively conjugated via oxime ligationto a therapeutic peptide mimetic containing anε-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acidexpression in the methylotrophic yeast was systematically optimized bymodulation of aaRS levels to express mutant human serum albumin inexcess of 150 mg/L in shake flasks, more than an order of magnitudebetter than that reported in S. cerevisiae. This methodology shouldallow the production of high yields of complex proteins with unnaturalamino acids whose expression is not practical in existing systems.

Recently, a methodology was developed that makes it possible togenetically encode a wide variety of unnatural amino acids with novelproperties (including fluorophores, metal ion chelators, photocaged andphotocrosslinking groups, NMR, crystallographic and IR probes, andpost-translationally modified amino acids) in both prokaryotic andeukaryotic organisms (Xie & Schultz (2006) “A chemical toolkit forproteins—an expanded genetic code.” Nat Rev Mol Cell Biol 7: 775-782;Chin et al. (2003) “An expanded eukaryotic genetic code.” Science 301:964-967; Wang et al. (2006) “Expanding the genetic code.” Annu RevBiophys Biomol Struct 35: 225-249). This is accomplished through theevolution of an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA(aaRS/tRNA_(CUA)) pair, designed to selectively insert a desiredunnatural amino acid in response to a nonsense or frameshift codon. Thusfar, this methodology has been employed to add more than 40 unnaturalamino acids to the genetic repertoires of Escherichia coli,Saccharomyces cerevisiae, and several lines of mammalian cells (Chin etal. (2003) “An expanded eukaryotic genetic code.” Science 301: 964-967;Wang et al. (2006) “Expanding the genetic code.” Annu Rev Biophys BiomolStruct 35: 225-249; Liu et al. (2007) “Genetic incorporation ofunnatural amino acids into proteins in mammalian cells.” Nat Methods 4:239-244). Orthogonality in these systems is achieved by transplanting anorthogonal aaRS/tRNA_(CUA) pair with distinct tRNA identity elementsinto the host organism such that no cross-aminoacylation occurs betweenthe host aminoacylation machinery and the transplanted aaRS/tRNA pair(while still maintaining function in translation). In the currentsystems, this has proven most successful using aaRS/tRNA_(CUA) pairsderived from the Methanococcus jannaschii tyrosyl-RS/tRNA_(CUA) pair inE. coli (Wang et al. (2001) “A general approach for the generation oforthogonal tRNAs.” Chem Biol 8: 883-890) and the E. coli tyrosyl- orleucyl-RS/tRNA_(CUA) pairs in S. cerevisiae (Chin et al. (2003) “Anexpanded eukaryotic genetic code.” Science 301: 964-967; Chin et al.(2003) “Progress toward an expanded eukaryotic genetic code.” Chem Biol10: 511-519) or mammalian cells (Liu et al. (2007) “Geneticincorporation of unnatural amino acids into proteins in mammaliancells.” Nat Methods 4: 239-244). Directed evolution is then used toalter the specificity of the orthogonal aaRS so that it recognizes theunnatural amino acid of interest and not an endogenous amino acid.

To apply this methodology for the production of large quantities ofproteins that are not easily expressed in bacterial hosts, a recombinantsystem is desired with low cost, scalability, and the ability to producecomplex, post-translationally modified proteins. One such host is Pichiapastoris, which is capable of producing mammalian proteins in yieldscomparable to those of E. coli (Cereghino et al. (2000) “Heterologousprotein expression in the methylotrophic yeast Pichia pastoris.” FEMSMicrobiol Rev 24: 45-66). Therapeutic proteins such as tumor necrosisfactor (TNF), tetanus toxin fragment C (TTC), and human serum albumin(HSA) have afforded expression levels >10 g L⁻¹ in high densityfermentations (Shekhar (2008) “Pichia power: India's biotech industryputs unconventional yeast to work.” Chem Biol 15: 201-202; Clare et al.(1991) “High-level expression of tetanus toxin fragment C in Pichiapastoris strains containing multiple tandem integrations of the gene.”Biotechnology (New York) 9: 455-460; Ohya et al. (2005) Optimization ofhuman serum albumin production in methylotrophic yeast Pichia pastorisby repeated fed-batch fermentation.” Biotechnol Bioeng 90: 876-887;Sreekrishna et al. (1989) “High-level expression, purification, andcharacterization of recombinant human tumor necrosis factor synthesizedin the methylotrophic yeast Pichia pastoris.” Biochemistry 28:4117-4125). P. pastoris' ability to produce proteins in such yields isattributed to its alcohol oxidase 1 promoter (P_(AOX1)), one of the mosthighly regulated and strongest promoters known (Cos et al. (2006)“Operational strategies, monitoring and control of heterologous proteinproduction in the methylotrophic yeast Pichia pastoris under differentpromoters: a review.” Microb Cell Fact 5: 17). In addition, P. pastorislacks endotoxins which can contaminate therapeutic proteins expressed inE. coli, and does not produce antigenic α1,3 glycan linkages as does S.cerevisiae (Cregg et al. (1993) “Recent advances in the expression offoreign genes in Pichia pastoris.” Biotechnology (New York) 11:905-910). Additionally, it is now possible to modulate glycosylationpatterns in P. pastoris, including control of sialylation (Li et al.(2006) “Optimization of humanized IgGs in glycoengineered Pichiapastoris.” Nat Biotechnol 24: 210-215). For these reasons, we undertookthe development of methodology to allow unnatural amino acids to begenetically encoded in P. pastoris. Here we report that eight unnaturalamino acids were site-specifically introduced into recombinant humanserum albumin (rHSA) expressed in this host with high yields andfidelities.

Materials

DNA primers used to perform the experiments described herein (e.g.,those listed in Table 1 and below) were purchased from Integrated DNATechnology (San Diego, Calif.). Restriction enzymes used to prepareconstructs described below were purchased from New England Biolabs(Beverly, Mass.). The pPIC3.5 k vector (map available athttp://tools.invitrogen.com/content/sfs/manuals/ppic3.5 kpao_man.pdf)and protocols for yeast competency, transformation, and media recipeswere purchased from Invitrogen (Carlsbad, Calif.). Multi-Copy PichiaExpression Kit Version F manual (Invitrogen Life, T., Vol. K1750-01,Edn. F 85 (Invitrogen Life Technologies, Carlsbad, Calif. 92008; 2005))is available athttp://tools.invitrogen.com/content/sfs/manuals/pichmulti_man.pdf. DNAwas amplified in E. coli DH10B (Invitrogen) or, when noted, by PCR usingplatinum pfx (Invitrogen). The rHSA gene (accession BC034023) wasobtained from the Mammalian Gene Collection (National Institutes ofHealth, Bethesda, Md.). All DNA constructs were confirmed by DNAsequencing (Genomics Institute of the Novartis Research Foundation, LaJolla, Calif.). The double auxotrophic Pichia pastoris strain, GS200(his4, arg4), and the pBLARG vector were gracious gifts from the JamesCregg laboratory at the Keck Graduate Institute, Claremont, Calif.Transformations of P. pastoris and E. coli were carried out on aGenePulser Xcell (Bio-Rad, Hercules, Calif.) using 2 and 1 mmelectroporation cuvettes (Fisher Scientific, Rochester, N.Y.).Tris-glycine (4-20%) SDS-PAGE gels for protein analysis were purchasedfrom Invitrogen. RNA was harvested via the protocols and reagentsaccompanying the Purelink miRNA isolation kit (Invitrogen) orRibo-pure-yeast kit (Ambion, Austin, Tex.). All relative gel banddensities were determined using Photoshop CS2 (Adobe, San Jose, Calif.).

Design of a Two Gene Cassette Expression System

Due to the relative instability of autonomously replicating plasmids inP. pastoris (Higgins et al. (1998) “Introduction to Pichia pastoris.”Methods Mol Biol 103: 1-15), a system was devised in which the targetgene of interest (e.g., rHSA) and the aaRS/tRNA_(CUA) pair were encodedin cassettes on two separate plasmids and stably integrated into thegenome. FIG. 1 provides vectors that were used to construct thecassettes for amber suppression in P. pastoris. Selectable markers oneach plasmid are indicated by the white arrows. Replication origins areindicated by black arrows. Promoters and transcriptional terminatorelements are indicated by vertically striped arrows. The doubleauxotroph P. pastoris strain GS200 (arg4, his4) was used as the hoststrain for protein expression, and the gene of interest, e.g., HSA, wasinserted into the commercially available pPIC3.5 k plasmid (HIS4,Gen^(R)) (FIG. 1 a) (Invitrogen Life, T., Vol. K1750-01, Edn. F 85(Invitrogen Life Technologies, Carlsbad, Calif. 92008; 2005)).

rHSA was used as a model protein given its utility in producing fusionproteins or peptide bioconjugates that enhance the serum half-life ofshort lived therapeutic polypeptides (Kim et al. (2003) “Development andcharacterization of a glucagon-like peptide 1-albumin conjugate: theability to activate the glucagon-like peptide 1 receptor in vivo.”Diabetes 52: 751-759; Huang et al. (2008) “Preparation andcharacterization of a novel exendin-4 human serum albumin fusion proteinexpressed in Pichia pastoris.” J Pept Sci 14: 588-595; Chuang et al.(2002) “Pharmaceutical strategies utilizing recombinant human serumalbumin.” Pharm Res 19: 569-577). Expression of rHSA in E. coli and S.cerevisiae is not practical due to the protein's complex disulfidecrosslinkages. However, such post-translational modifications can bemade in P. pastoris. Additionally, the 24 amino acid mammalian “pre-pro”leader sequence of HSA (FIG. 1 e) is fully compatible with expression inP. pastoris and allows export of the mature protein into the media(Kobayashi (2006) “Summary of recombinant human serum albumindevelopment.” Biologicals 34: 55-59). The pre-pro leader peptide iscleaved during transport to yield the mature protein (e.g.,rHSA_(E37X)). The 37^(th) residue in rHSA, relative to SEQ ID NO: 1denotes the unnatural amino acid incorporated in response to the ambercodon.

First, cassettes encoding rHSA_(WT) and rHSA_(E37X) that could be usedto transform GS200 were prepared. The pPIC3.5K-rHSA (wild type rHSA)construct was prepared as follows: The wild type rHSA gene was obtainedfrom the Mammalian Gene Collection (NIH), gene accession BC₀₃₄₀₂₃. Forcompatibility with pPIC3.5 k linearization (Invitrogen Life, T., Vol.K1750-01, Edn. F 85 (Invitrogen Life Technologies, Carlsbad, Calif.92008; 2005)), BglII sites were removed from rHSA by a modified QuikChange mutagenesis (Stratagene) protocol (Zheng et al. (2004) “Anefficient one-step site-directed and site-saturation mutagenesisprotocol.” Nucleic Acids Res 32: e115) using primers (IDT): -BglII 1F,5′-GAC AGA CCT TAC CAA AGT CCA CAC GGA ATG CTG CCA TG-3′ and -BglII 1R,5′-GGT AAG GTC TGT CAC TAA CTT GGA AAC TTC TGC AAA CTC AGC TTT GGG-3′for BglII₇₈₁, and -BglII 2F, 5′-CAT GGA GAC CTG CTT GAA TGT GCT GAT GACAGG GCG G-3′ and -BglII 2R, 5′-CAA GCA GGT CTC CAT GGC AGC ATT CCG TGTGGA C-3′ for BglII₈₁₇ to create rHSA_(WT). rHSA_(WT) was amplified usingprimers: HSA Forward, 5′-ATC CGA GGA TCC AAA CGA TGA AGT GGG TAA CCT TTATTT CCC TTC TIT TTC-3′ and HSA Reverse, 5′-GCT AAC GAA TTC ATT ATA AGCCTA AGG CAG CTT GAC TTG CAG C-3′, digested with EcoRI and BamHI (NEB)and ligated into the similarly digested pPIC3.5 k vector (Invitrogen,vector map available athttp://tools.invitrogen.com/content/sfs/manuals/ppic3.5 kpao_man.pdf) tocreate pPIC3.5 k-rHSA_(WT) (or pPIC3.5 k-rHSA_(E37X), as describedbelow). Constructs were confirmed by DNA sequencing and amplified in E.coli DH10B (Invitrogen).

The Glu37TAG mutant rHSA (rHSA_(E37X)) construct was generated by PCRmutagenesis. The Glu37 codon was replaced by the amber codon TAG usingthe modified Quik Change protocol and the primers: Glu37 F′, 5′-GAT TGCCTT TGC TCA GTA TCT TCA GCA GTG TCC ATT TTA GGA TCA T-3′ and Glu37 R′,5′-GTT TTT GCA AAT TCA GTT ACT TCA ITC ACT AAT TTT ACA TGA TCC TAA AATGG-3′. Glu37 is in a solvent accessible helix which should facilitatethe conjugation of peptides to a chemically reactive unnatural aminoacid (i.e., p-acetylphenylalanine, FIG. 6b, Structure 1) introduced atthis site, and ensure that incorporation of relatively bulky groupsminimally disrupt native protein structure and folding. rHSA_(E37X) wasthen amplified using primers: HSA Forward, 5′-ATC CGA GGA TCC AAA CGATGA AGT GGG TAA CCT TTA TTT CCC TTC TTT TTC-3′ and HSA Reverse, 5′-GCTAAC GAA TTC ATT ATA AGC CTA AGG CAG CTT GAC TTG CAG C-3′, digested withEcoRI and BamHI (NEB) and ligated into the similarly digested pPIC3.5 kvector, as described above for HSA_(WT), placing HSA_(E37X) under thetranscriptional control of the AOX1 promoter and terminator. TherHSA_(E37X)construct, pPIC3.5 k-rHSA_(E37X), was confirmed by DNAsequencing, as described above.

Linearization of pPIC3.5 k-rHSA_(E37X) or pPIC3.5 k-rHSA_(WT) in the 5′AOX1 promoter allows genomic integration of one or more copies of thetransformed cassette; generally more copies result in higher overallyields of target protein (Buckholz et al. (1991) “Yeast systems for thecommercial production of heterologous proteins.” Biotechnology (N Y) 9:1067-1072). Integration in this manner leaves the AOX1 gene intact,retaining the yeast's ability to rapidly utilize methanol (Mut⁺phenotype). Alternatively, gene replacement can be carried out bylinearization on either side of AOX1 gene, resulting in replacement ofthe AOX1 gene by the pPIC3.5 k vector (Invitrogen Life, T., Vol.K1750-01, Edn. F 85 (Invitrogen Life Technologies, Carlsbad, Calif.92008; 2005)). Yeast lacking AOX1 rely on the weaker AOX2 gene formethanol utilization and are phenotypically mut^(s). Because expressionof rHSA is commonly carried out with mut^(s) yeast (Kupcsulik et al.(2005) “Optimization of specific product formation rate by statisticaland formal kinetic model descriptions of an HSA producing Pichiapastoris Mut(S) strain.” Chem Biochem Eng Q 19: 99-108), pPIC3.5 kHSA_(E37X) and pPIC3.5 k-rHSA_(WT) were linearized and used to replacethe AOX1 gene to yield GS200-rHSA_(E37X) or GS200-rHSA_(WT) (bothstrains are HIS4, arg4, Gen^(R), mut^(s)). Successful transformants grewnormally on minimal media plates lacking histidine and on rich mediaplates containing up to 0.25 mg/mL of the aminoglycoside antibioticGeneticin®.

To transform GS200 with a pPIC3.5 k HSA_(E37X) or pPIC3.5 k-rHSA_(WT)cassette, 20 μg of pPIC3.5 k-rHSA_(E37X) or pPIC3.5 k-rHSA_(WT) waslinearized with BglII (NEB), concentrated to 10 μl by ethanolprecipitation, added to 80 μl of freshly competent GS200 in a 2 mmelectroporation cuvette (Fisher), and electroporated with the P.pastoris settings (2000 V, 25 μF, 200Ω) on a GenePulser Xcell (BioRad).Cells were recovered in 1 ml cold 1 M sorbitol. 250 μl of recoveredcells was plated on regeneration dextrose Bacto agar (RDB) plates (15cm) supplemented with 4 mg ml⁻¹ L-arginine (arg) and incubated at 30° C.After 3 days, colonies were picked into 96 well 2 ml blocks (Nunc) with1 ml yeast peptone dextrose (YPD) media and grown overnight (29.2° C.,300 r.p.m.). The cultures were diluted 1:100 and 1-2 μl replica platedon YPD agar plates containing 0.25 mg ml⁻¹ Geneticin® (Invitrogen) andincubated at 30° C. After 4 days, GS200-rHSA_(E37X) transformant G3showed good growth, was picked, and made competent. A GS200-rHSA_(WT)clone that showed good growth was also picked.

To integrate the orthogonal aaRS/tRNA_(CUA) pair into the genome, thepreviously developed pPR1-P_(PGK1)+3SUP4-tRNA_(CUA) ^(Tyr) vector (Chenet al. (2007) “An improved system for the generation and analysis ofmutant proteins containing unnatural amino acids in Saccharomycescerevisiae.” J Mol Biol 371: 112-122) (FIG. 1 b) for optimized ambersuppression and recombinant over-expression in S. cerevisiae wasmodified. The p-acetylphenylalanine-(pApa, FIG. 6 b, Structure 1)specific aminoacyl-tRNA synthetase (pApaRS), previously evolved in S.cerevisiae (Zhang et al. (2003) “A new strategy for the site-specificmodification of proteins in vivo.” Biochemistry 42, 6735-6746), wasinserted between the alcohol dehydrogenase 1 promoter (P_(ADH1)) andterminator (T_(ADH1)) with a His₆-tag to assay its expression.

To prepare the aaRS/tRNA_(CUA) pair for integration into the P. pastorisgenome, the pPR1-P_(PGK1)+3SUP4-tRNA_(CUA) ^(tyr) vector (Chen et al.(2007) “An improved system for the generation and analysis of mutantproteins containing unnatural amino acids in Saccharomyces cerevisiae.”J Mol Biol 371: 112-122) harboring the pApaRS was amplified by PCR,excluding the TRP and 2μ origin regions, to add restriction sites KpnIand HindIII with primers: pESC F, 5′-TAC CAC TAG AAG CTT GGA GAA AAT ACCGCA TCA GGA AAT TGT AAA CGT-3′ and pESC R, 5′-GTG AGG GCA GGT ACC GTTCTG TAA AAA TGC AGC TCA GAT TCT TTG TTT G-3′ and digested with HindIIIand KpnI (NEB). The ARG4 coding region was amplified from pBLARG (giftfrom the James Cregg laboratory, Keck Graduate Institute, Claremont,Calif.) with primers: ARG4 F new, 5′-AAA TAT GGT ACC TGC CCT CAC GGT GGTTAC GGT-3′ and ARG4 R new, 5′-CAT TTC AAG CTT CTA GTG GTA GGA ATT CTGTAC CGG TTT AC-3′, digested with KpnI and HindIII, and ligated into thesimilarly digested pPR1-P_(PGK1)+3SUP4-tRNA_(CUA) ^(tyr) PCR product tocreate the recombinant eukaryotic ARG4 vector, pREAV-P_(ADH1)-pApaRS.The pREAV-P_(ADH1)-pApaRS was amplified by PCR, excluding theP_(ADH1)-pApaRS-T_(ADH1) region, to add restriction sites AscI and AflIIwith primers: pESC-AOX-KETO F, 5′-ATC GTA CTT AAG GAA AGC GTA CTC AAACAG ACA ACC ATT TCC-3′ and pESC-AOX-KETO R, 5′-TTC TCA GGC GCG CCA TCGCCC TTC CCA ACA GTT GCG-3′. Constructs were confirmed by size mappingand sequencing.

The cognate E. coli tRNA_(CUA) ^(tyr) lacking the 5′ CCA was inserted asthree tandem repeats behind the phosphoglycerate kinase 1 promoter(P_(PGK1)). To aid in posttranscriptional processing, the tRNAs wereflanked by regions from the yeast suppressor tRNA gene, SUP4, aspreviously described (Chen et al. (2007) “An improved system for thegeneration and analysis of mutant proteins containing unnatural aminoacids in Saccharomyces cerevisiae.” J Mol Biol 371: 112-122). Eukaryoticdownstream processing adds the 5′ CCA that is required for tRNAfunction. The 2μ origin and phosphoribosyl anthranilate isolmerase (TRP)marker of pPR1-P_(PGK1)+3SUP4-tRNA_(CUA) ^(tyr) were replaced by thearginosuccinate lyase (ARG4) coding region to give the recombinanteukaryotic ARG4 vector (pREAV-P_(ADH1)-pApaRS) (FIG. 1 c). Propagationof this cassette is only possible in the event of genomic incorporationsince it lacks a eukaryotic origin of replication. Transformations tocreate GS200-rHSA_(E37X)/pREAV-P_(ADH1)-pApaRS were carried out usingthe protocol described above with competent G3 (e.g., competentGS200-rHSA_(E37X) transformant), except recovered cells were plated onRDB plates lacking L-histidine and arginine. After 3 days colonies werepicked into 96 well 2 ml blocks and rescreened as above for resistanceto 0.25 mg ml⁻¹ Geneticin®. GS200-rHSA_(WT)/pREAV P_(ADH1)-pApaRS (HIS4,ARG4, Gen^(R), mut^(s)) was created in identical fashion to isolatecolony F2. Thus, linearization of pREAV-P_(ADH1)-pApaRS in the ARG4coding region, and subsequent transformation into GS200-HSA_(E37X) andGS200-HSA_(WT) gave the fully prototrophic P. pastorisGS200-HSA_(E37X)/pREAV-P_(ADH1)-pApaRS andGS200-HSA_(WT)/pREAV-P_(ADH1)-pApaRS strains, respectively. (Bothstrains are HIS4, ARG4, Gen^(R), mut^(s)).

Amber Suppression in P. pastoris.

All protein expression experiments followed protocols for mut^(s) foundin the Multi-Copy Pichia Expression Kit (Invitrogen Life, T., Vol.K1750-01, Edn. F 85 (Invitrogen Life Technologies, Carlsbad, Calif.92008; 2005)). 14 colonies for GS200-rHSA_(E37X)/pREAV-P_(ADH1)-pApaRSor GS200-rHSA_(WT)/pREAV-P_(ADH1)-pApaRS were picked from platescontaining 0.25 mg ml⁻¹ Geneticin® and grown to near saturation(OD₆₀₀≈12-18) in 10 ml buffered glycerol-complex medium (BMGY) (29.2°C., 300 r.p.m.). Cultures were centrifuged at 1500 g (10 min), andresuspended in 2 ml buffered methanol-complex media (BMMY) with 2 mMpApa amino acid (SynChem, Des Plaines, Ill.). Growth was continued for 6days, with methanol supplementation to 0.5% every 24 hrs. 200 μl (10%culture volume) of media or sterile water was added every 24 hrs toaccount for evaporation. 50 μl of media was removed every 24 hrs andcleared of cells by centrifugation at 3000 g (5 min). 25 μl of thecleared media was added to 12.5 μl of SDS loading buffer, heated for 1min at 95° C., and run on a SDS-PAGE gel (Invitrogen) (150 V 1 h).

Amber suppression only occurs in yeast harboring both vectors grown withmethanol and pApa amino acid (pApa AA). Clones isolated fromGS200-HSA_(wT)/pREAV-P_(ADH1)-pApaRS produced full length rHSA_(WT)(66.5 kDa) visible by Coomassie stain (40% methanol, 10% acetic acid,50% water, 0.1% (w/v) Coomassie Brilliant Blue R250 (Sigma-Aldrich)) ona sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE)gel after two to three days when grown under methanol inducingconditions. The expression of rHSA_(WT) peaked after 6 days. Clone F2-wtfor GS200-rHSA_(WT)/pREAV-P_(ADH1)-pApaRS showed highest expression andwas used in further comparisons. In contrast, clones fromGS200-HSA_(E37X)/pREAV-P_(ADH1)-pApaRS failed to produce full lengthrHSA_(E37pApa) when grown for six days with methanol as the primarycarbon source and pApa amino acid supplementation. (See, e.g., FIG. 2 b;lane 2 is GS200; lane 3 is GS200-HSA_(E37X); lane 4 isGS200-pREAV-P_(AOX1)-pApaRS; lanes 5-7 areGS200-HSA_(E37X)/pREAV-P_(AOX1)-pApaRS; and lane 8 isGS200-HSA_(WT)/pREAV-P_(ADH1)-pApaRS.

Genomic integration of all constructs was confirmed by genomic PCR (FIG.7; four clones were chosen from one transformation and labeled 1-4).Expected PCR products were rHSA (1851 bp), pApaRS (1317 bp), and tRNAcassette (1100 bp). The lack of pApaRA amplification in clone 2 islikely a technical artifact. The bottom gel of FIG. 2 a depicts theresults of a northern blot performed to assay tRNA_(cuA) expression inS. cerevisiae+pPR1-P_(PGK1)-3SUP4-tRNA (lane 1) and P.pastoris+pREAV-P_(ADH1)-pApaRS (lane 2). For a negative control, lanes 3and 4 are S. cerevisiae and P. pastoris strains lacking vectors,respectively. The top gel of FIG. 2 a shows a northern blot for theendogenous serine tRNA and illustrates equal miRNA preparation in allsamples.

Briefly, northern blots to confirm transcription of the tRNA_(CUA) wereperformed as follows: two P. pastoris clones, G3-2 and GS200, and two S.cerevisiae clones, SCY4-pPR1-P_(PGK1)+2SUP4-tRNA, and SCY4, were grownunder their respective expression conditions, and micro RNA (miRNA)harvested. 2 μg of RNA from each sample was loaded onto two 6% NovexTBE-Urea gel (Invitrogen), and run at 180 V for 1 h. RNA was transferredto a Biodyne B nylon membrane (Pall Life Science) using an XCellsurelock mini-cell (Invitrogen) in 0.5×TBE buffer (Invitrogen) andaccompanying protocols. The membranes were auto cross-linked with UVStratalinker 2400 (Stratagene, La Jolla, Calif.). Hybridization anddetection was carried out with protocols and reagents found in theNorth2South chemiluminescent hybridization and detection kit (Pierce,Rockford, Ill.). One blot was incubated with biotinylated probesspecific for tRNA^(ser): tRNAser cere 1,5′-/5Biosg/CAT TTC AAG ACT GTCGCC TTA ACC ACT CGG CCA T-3′, tRNAser cere 2,5′-/5Biosg/GAA CCA GCG CGGGCA GAG CCC AAC ACA TTT CAA G-3′, tRNAser pitch 1,5′-/5Biosg/CTG CAT CCTTCG CCT TAA CCA CTC GGC CAT CGT A-3′, tRNAser pich 2,5′-/5Biosg/ACA CGAGCA GGG TTC GAA CCT GCG CGG GCA GAG C-3′ and the second blot incubatedwith biotinylated probes specific for tRNA_(CUA) ^(tyr): tRNA 5′ biot,5′-/5Biosg/GGA AGG ATT CGA ACC TTC GAA GTC GAT GAC GG-3′ and tRNA 3′biot, 5′-/5Biosg/TCT GCT CCC TIT GGC CGC TCG GGA ACC CCA CC-3′. Probeswere incubated overnight at 55° C., bound to a streptavidin-horseradishperoxidase (HRP) conjugate, and detected with a luminol/enhancer—stableperoxide solution (Pierce) (FIG. 2 a). Relative tRNA amounts weredetermined by band density. The results of FIG. 2 indicate thattranscription of the tRNA_(CUA) was found to be approximately 1.5 timesgreater in P. pastoris+pREAV-P_(ADH1)-pApaRS than the same cassette inS. cerevisiae by northern blot analysis (FIG. 2 a).

Despite these results, no pApaRS was detectable by western blot for theHis_(6x)-tag in the GS200-HSA_(E37X)/pREAV-P_(ADH1)-pApaRS strain (FIG.8). Four separate clones from a single transformation were tested. Theseresults indicated that the lack of amber suppression was linked to poorincorporation of the pApaRS. (Western blots were performed as describedelsewhere herein.)

To address the poor expression of pApaRS, pREAV was modified to driveexpression of pApaRS with the powerful P_(AOX1) promoter, and pApaRSexpression was further enhanced by adding a Kozak consensus sequence(ACCATGG) (Kozak (1990) “Downstream secondary structure facilitatesrecognition of initiator codons by eukaryotic ribosomes.” Proc Natl AcadSci USA 87: 8301-8305) to the 5′ end of the pApaRS gene. The ADH1terminator (T_(ADH1)) was also replaced by the AOX1 terminator(T_(AOX1)) in the final construct pREAV-P_(AOX1)-pApaRS (FIG. 1 d). Tocreate pREAV-P_(AOX1)-pApaRS, the AOX1 promoter and terminator sequenceswere derived from pPIC3.5 k. The pApaRS was amplified with primers:KETO-Koz-F, 5′-TTC TGA GAA TTC ACC ATG GCA AGC AGT AAC TTG AU AAA CAATTG C-3′ and KetoRS R 6×His, 5′-TAG GCT CGG CCG CTT AGT GGT GGT GGT GGTGGT GTT TCC AGC AAA TCA GAC AGT AAT TCT ITT TAC-3′, digested with EcoRIand NotI (NEB) and ligated into the similarly digested pPIC3.5 k tocreate pPIC3.5 k-pApaRS. The P_(AOX1)-pApaRS-T_(AOX1) coding region wasamplified from pPIC3.5 k-pApaRS with primers: pPIC-keto AOX5 F, 5′-ATCGTA CTT AAG AGA TCT AAC ATC CAA AGA CGA AAG GTT GAA TGA AAC-3′ andpPIC-keto AOXTT R, 5′-TGC ACA GGC GCG CCA AGC TTG CAC AAA CGA ACT TCTCAC TTA ATC ITC-3′, digested with AscI and AflII (NEB) and ligated intothe similarly digested pREAV-P_(ADH1)-pApaRS PCR product to createpREAV-P_(AOX1)-pApaRS. Constructs were confirmed by size mapping andsequencing, as described above.

The GS200-pREAV-P_(AOX1)-pApaRS (his4, ARG4, Gen^(R), mut^(s)) strainwas constructed by transforming pREAV-P_(AOX1)-pApaRS into GS200 butplated on RDB plates supplemented with 4 mg ml⁻¹ histidine and notfurther screened for Geneticin® resistance. Transformations to createGS200-rHSA_(E37X)/pREAV-P_(AOX1)-pApaRS (HIS4, ARG4, Gen^(R), mut^(s))were carried out using the protocol described above with competent G3(e.g., GS200-rHSA_(E37X)) except recovered cells were plated on RDBplates lacking L-histidine (His) and Arg. Clones from thistransformation produced full length rHSA_(E37pApa) only in the presenceof methanol and pApa amino acid, at levels approximately 10-20% ofidentical clones harboring rHSA_(WT). Protein was visible by SDS-PAGEgel two to three days post methanol induction, and peaked six days afterexpression with methanol supplementation to 0.5% every 24 h (FIG. 2 b).Yeast lacking the pREAV cassette, pPIC3.5 k cassette, methanolsupplementation, or pApa amino acid failed to produce protein detectableby Coomassie staining. The lack of protein expression in the absence ofpApa amino acid indicates that no cross aminoacylation occurs betweenthe pApaRS/tRNA_(CUA) ^(tyr) pair and the endogenous aminoacylationmachinery. Site-specific incorporation of pApa into rHSA_(E37X) wasconfirmed by tryptic digest, LC-MS/MS (FIG. 2 c). pApa (denoted E* inFIG. 2 c) was incorporated at residue 37 of mature rHSA_(E37pApa). Thesubstitution is supported without ambiguity by the observed fragment ionseries.

To express enough rHSA_(E37pApa) protein to perform the LC-MS/MSanalysis, the protein expression analysis conditions described abovewere modified. Briefly, 1 L of BMGY was inoculated with 20 ml ofsaturated G3-2 culture in YPD and grown (˜24 h, 29.2° C., 300 r.p.m.) toOD₆₀₀≈12-18. The culture was centrifuged at 1500 g, and resuspended in200 ml buffered minimal methanol (BMM) supplemented with 10% BMMY and 2mM pApa. After 6 days of growth (29.2° C., 300 r.p.m. with methanol andvolume supplementation) the culture was centrifuged at 3000 g, cellsdiscarded, and supernatant passed through a 0.22 μm filter (Milipore,Billerica, Mass.). The supernatant was ammonium sulfate (NH₄SO)precipitated by addition of NH₄SO₄ with slow stirring at 4° C. to 50% ofsaturation (58.2 g), centrifugation at 20,000 g for 20 min, and again byaddition of NH₄SO₄ to 75% of saturation (31.8 g), and centrifugation at20,000 g for 20 min. The second precipitation contained rHSA_(E37pApa)and was resuspended in FPLC Buffer A (25 mM Tris-HCl, 25 mM sodiumchloride, 1 mM EDTA, 1× protease inhibitor cocktail (Roche, Basel, CH),pH=8.5). The resolubilized protein was purified with MonoQ 5/5 column(GE Healthcare) on an AKTA purifier FPLC (Amersham Biosciences,Piscataway, N.J.) (elution at 20-35% Buffer B (Buffer A+1 M NaCl)).Fractions were analyzed by SDS-PAGE gel, combined, dialyzed with a 30MWCO dialysis cassette (Pierce) to PBS, and purified with a Superdex 20010/300 GL (GE Healthcare) on an AKTA purifier FPLC (elution after 14 minin PBS at 0.5 ml min⁻¹). Fractions were analyzed by SDS-PAGE gel,combined, and purified with a C8 Vydac HPLC column (300 mm, 200 Å, 5 μm,Grace) on a Dynamax HPLC (Rainin, Oakland) (elution at 40-46% MeCN inwater, 0.1% TFA). Fractions were analyzed by SDS-PAGE gel, andrHSA_(E37pApa)-containing fractions were flash frozen and lyophilized toa white powder. Purification of rHSA_(WT) from F2-wt was done in similarfashion.

To perform the tryptic digest and nano-RP LC-MS/MS, purified rHSA_(E37X)was digested overnight with trypsin under reducing conditions (10 mMTCEP, 1M guanidine HCl, 100 mM triethanolamine HCl, pH=7.8). The digestwas purified by reversed-phase solid-phase extraction with a Sep-Pak,C18, (Waters, Milford, Mass.) and lyophilized. Oxidation of cysteines tocysteic acid and methionine to methionine sulfone was performed byincubation of lyophilized peptides with performic acid (9 partsconcentrated formic acid+1 part 30% H₂O₂) (Matthiesen et al. (2004) “Useof performic acid oxidation to expand the mass distribution of trypticpeptides.” Analytical chemistry 76, 6848-6852) for 1 h on ice. Thereaction was quenched by addition of an excess of mercaptoethanol and20× dilution with water. Nano-RP LC-MS/MS was performed with a HPLCsystem (Agilent Technologies, Santa Clara, Calif.) equipped with an LTQOrbitrap hybrid mass spectrometer (ThermoElectron, Rochester, N.Y.).Tryptic digests were loaded onto the precolumn (4 cm, 100 μm i.d., 5 μm,Monitor C18, Column Engineering, Chicago, Ill.) of a vented column setup(Licklider et al. (2002) “Automation of nanoscale microcapillary liquidchromatography-tandem mass spectrometry with a vented column.”Analytical chemistry 74: 3076-3083) at a flow rate of ˜2 μl min⁻¹. Aftera load/wash period of 10 min gradient elution was started by switchingthe precolumn in line with the analytical column (10 cm, 75 μm i.d., 5μm C18). The chromatographic profile was from 100% solvent A (0.1%aqueous acetic acid) to 50% solvent B (0.1% acetic acid in acetonitrile)in 40 min at ˜100 nl min⁻¹. Data-dependent MS/MS acquisitions wereperformed following a top 10 scheme in which the mass spectrometer wasprogrammed to first record a high-resolution Orbitrap scan (m/z500-2,000) followed by 10 data-dependent MS/MS scans (relative collisionenergy=35%; 3 Da isolation window). The raw data was searched againstthe SwissProt 51.6 database using MASCOT (Matrixscience, London, UK) forprotein identification with pApa as a variable modification.

Optimization of Expression

In an effort to optimize expression of rHSA_(E37pApa), aGS200-rHSA_(E37X)/pREAV-P_(AOX1)-pApaRS (HIS4, ARG4, Gen^(R)) fastmethanol utilization (Mut⁺) mutant was created by insertion of pPIC3.5k-rHSA_(E37X) into the region 5′ of the AOX1 gene locus (this retainsthe integrity of the AOX1 gene). Genomic insertion in this manner canlead to multimerization, yielding tandem copies of both the Geneticin®resistance marker and the gene of interest (Cereghino et al. (2000)“Heterologous protein expression in the methylotrophic yeast Pichiapastoris.” FEMS Microbiol Rev 24: 45-66). To createGS200-rHSA_(E37X)/pREAV-P_(AOX1)-pApaRS (HIS4, ARG4, Gen^(R), Mut), 20μg of pPIC3.5 k-rHSA_(E37X) was linearized with SacI or SalI (NEB) andtransformed into freshly competent GS200 as previously described. Cellswere recovered in 1 ml of cold 1 M sorbitol, and plated on RDB platessupplemented with 0.4 mg ml⁻¹ arginine. Colonies were picked into a 2 ml96 well block with 1 ml YPD, grown to saturation (29.2° C., 300 r.p.m.),diluted 1:100, and replica plated on plates containing 0 to 3.0 mg ml⁻¹Geneticin®. Clone 1D12 which survived up to 1.0 mg ml⁻¹ Geneticin®, wasmade competent, transformed with pREAV-P_(AOX1)-pApaRS as previouslydescribed and plated on RDB plates lacking Arg and His. Colonies werepicked into a 1 ml 96 well block, grown to saturation, diluted 1:100,and rescreened on Geneticin® 1.0 mg ml⁻¹ plates. 14 surviving cloneswere picked, and tested for rHSA_(E37pApa) expression in the presence ofpApa amino acid and methanol. The mut^(s) protocol was used, asdescribed above. Clone K5 showed greatest protein expression, and wascompared to G3-2 in test expressions (FIG. 9). 25 μl of cleared mediafrom a GS200-rHSA_(E37X)/pREAV-P_(AOX1)-pApaRS (mut⁺) culture (FIG. 9,lane 1) and a GS200-rHSA_(E37X)/pREAV-P_(AOX1)pApaRS (mut^(s)) culture(FIG. 9, lane 2) were analyzed on an SDS-PAGE gel and stained withCoomassie. The resulting clone K5 displayed resistance to Geneticin® upto 1 mg ml⁻¹; whereas the aforementioned mut^(s) clone G3-2 died above0.25 mg ml⁻¹ Geneticin®, consistent with the incorporation of multiplecopies of the cassette (Cereghino et al. (2000) “Heterologous proteinexpression in the methylotrophic yeast Pichia pastoris.” FEMS MicrobiolRev 24: 45-66; Invitrogen Life Technologies, Carlsbad, Calif. 92008;2005). Analysis of full length rHSA_(E37pApa) expression from isolatedclones in the presence of methanol and pApa amino acid showed thatapproximately 1.5-2.0 times more protein was produced than with themut^(s) counterpart (FIG. 9). Relative amounts of protein determined byband density.

To further increase yields of rHSA_(E37pApa) six different promoters(including P_(AOX1)) were compared for their ability to drive pApaRStranscription in the pREAV vector. Transcript mRNA levels, pApaRSprotein levels, and overall rHSA_(E37pApa) yields were assayed. Twoconstitutive promoters derived from yeast, GTP binding protein I (YPT1)(Sears et al. (1998) “A versatile set of vectors for constitutive andregulated gene expression in Pichia pastoris.” Yeast 14: 783-790; Segevet al. (1988) “The yeast GTP-binding YPT1 protein and a mammaliancounterpart are associated with the secretion machinery.” Cell 52:915-924); glyceraldehyde-3-phosphate dehydrogenase (GAP) (Cos et al.(2006) “Operational strategies, monitoring and control of heterologousprotein production in the methylotrophic yeast Pichia pastoris underdifferent promoters: a review.” Microb Cell Fact 5: 17; Waterham et al.(1997) “Isolation of the Pichia pastoris glyceraldehyde-3-phosphatedehydrogenase gene and regulation and use of its promoter.” Gene 186:37-44); three methanol inducible promoters from alcohol oxidase II(AOX2) (Ohi et al. (1994) “The positive and negative cis-acting elementsfor methanol regulation in the Pichia pastoris AOX2 gene.” Mol Gen Genet243: 489-499), formaldehyde dehydrogenase I (FLD1) (Cos et al. (2006)“Operational strategies, monitoring and control of heterologous proteinproduction in the methylotrophic yeast Pichia pastoris under differentpromoters: a review.” Microb Cell Fact 5: 17); and isocitrate lyase I(ICL1) (Cos et al. (2006) “Operational strategies, monitoring andcontrol of heterologous protein production in the methylotrophic yeastPichia pastoris under different promoters: a review.” Microb Cell Fact5: 17) were chosen based on their compatibility with methanol induction.A truncated version of P_(AOX2) was used which enhances the promoter bydeleting one of the two repressor binding sequences (Ohi et al. (1994)“The positive and negative cis-acting elements for methanol regulationin the Pichia pastoris AOX2 gene.” Mol Gen Genet 243: 489-499). The useof the somewhat weaker P_(YPT1) and P_(GAP) promoters (Sears et al.(1998) “A versatile set of vectors for constitutive and regulated geneexpression in Pichia pastoris.” Yeast 14: 783-790) could be useful inthe event that overproduction of the synthetase is toxic to the yeast,or sequesters cellular energy away from production of rHSA_(E37X).

All promoters were amplified by PCR from P. pastoris genomic DNA alongwith their 5′ untranslated regions (FIG. 10). P_(AOX2), P_(YPT1),P_(ICL1), P_(FLD1), and P_(GAP) were separately amplified by PCR fromgenomic DNA (P. pastoris GS200) using the following primers: PAOX2 F,5′-GTA TCG CTT AAG TCC AAG ATA GGC TAT TTT TGT CGC ATA AAT TTT TGT C -3′and PAOX2 R, 5′-CGT TAG CCA TGG TTT TCT CAG TTG ATT TGT TTG TGG GGA TTTAGT AAG TCG-3′; PYPT1 F, 5′-GTA TCG CTT AAG CAT ATG ATG AGT CAC AAT CTGCTT CCA CAG ACG AG-3′ and PYPT1 R, 5′-CGT TAG CCA TGG GAC TGC TAT TATCTC TGT GTG TAT GTG TGT ATT GGG C-3′; PICL1 F, 5′-GTA TCG CTT AAG GAATTC GGA CAA ATG TGC TGT TCC GGT AGC TTG-3′ and PICL1 R, 5′-CGT TAG CCATGG TCT TGA TAT ACT TGA TAC TGT GTT CTT TGA ATT GAA AG-3′; PFLD1 F,5′-GTA TCG CTT AAG GCA TGC AGG AAT CTC TGG CAC GGT GCT AAT GG-3′ andPFLD1 R, 5′-CGT TAG CCA TGG TGT GAA TAT CAA GAA TTG TAT GAA CAA GCA AAGTTG G-3′; PGAP1 F, 5′-GTA TCG CTT AAG GGA TCC TTT TTT GTA GAA ATG TCTTGG TGT CCT CGT C-3′ and PGAP1 R, 5′-CGT TAG CCA TGG TGT GTT TTG ATA GTTGTT CAA TTG ATT GAA ATA GGG AC-3′; respectively. FIG. 10 shows anethidium bromide stained gel with a 1 kb+ ladder flanking the PCRproducts. The expected lengths of the PCR products are PAOX2 342 bp,PYPT1 508 bp, PICL1 683 bp, PFLD1 597 bp, and PGAP 493 bp. The PCRamplified fragments were digested with AftII and NcoI (NEB) and ligatedinto the similarly digested pREAV-P_(AOX1)-pApaRS (after removal of theP_(AOX2) coding region via agarose gel purification) to create thepREAV-P_(promoter)-pApaRS.

After sequence confirmation, each promoter was cloned into the pREAVvector 5′ of pApaRS in place of P_(AOX1), and transformed into theMut⁺GS200-HSA_(E37X) created previously. The terminator remainedT_(AOX1). FIG. 3 a provides a linear map of pREAV-P_(promoter)-pApaRSillustrating the promoter region being varied. Promoters were PCRamplified from genomic DNA (as in the experiments whose results aredepicted in FIG. 7). The plasmids (like the previously describedconstruct pREAV-P_(AOX1)-PApaRS) were linearized with AatII, transformedinto freshly competent GS200-rHSA_(E37X) (clone 1D12), and plated on RDBplates lacking Arg and His as previously described to createGS200-rHSA_(E37X)/pREAV-P_(promoter)-pApaRS (HIS4, ARG4, Gen^(R), Mut⁺).Surviving clones were screened for Geneticin® resistance at 0.75 and 1.0mg ml⁻¹. 48 clones corresponding to each promoter were picked into 1 mL96 well blocks containing BMGY and grown to saturation (29.2° C., 24 h,300 r.p.m.). The saturated cultures were centrifuged at 1500 g for 10min and cells were resuspended in 200 μL BMMY+2 mM pApa amino acid.After 6 days (29.2° C., 300 r.p.m., with supplementation), the media wascleared by centrifugation at 3000 g for 10 minutes, and 1-2 μL of thecleared media spotted on a 0.45 micron nitrocellulose membrane (Bio-Rad)using a 96 well pin tool. The membrane was probed with the HSA antibody[1A9] HRP conjugate (Abcam) using standard western blotting techniques(Burnette (1981) “Western blotting: electrophoretic transfer of proteinsfrom sodium dodecyl sulfate—polyacrylamide gels to unmodifiednitrocellulose and radiographic detection with antibody andradioiodinated protein A.” Anal Biochem 112: 195-203), and detected withECL HRP chemiluminescence detection reagents and protocols (GEHealthcare). The two highest expressing clones corresponding to eachpromoter (AOX2: A6 and B7; YPT1: D11 and B7; ICL1: E5, and H3; FLD1: E11and F3; GAP: B7 and B10; and AOX1: E3 and E7) were chosen for paralleltest expressions (FIGS. 3 and 4).

P-_(promoter)-pApaRS expression levels were monitored by northern andwestern blots after 6 days of methanol induction and compared toP_(AOX1)-pApaRS (FIG. 3 b). Due to inherent expression variability withP. pastoris, two clones were chosen for western blot analysis. The twoclones from each transformation of GS200-rHSA_(E37X) withpREAV-P_(promoter)-pApaRS were grown with methanol as the primary carbonsource for 6 days, lysed, separated on an SDS-PAGE gel (FIG. 3 b, topgel). The gel was stained with Coomassie to verify equal loading.Lysates were analyzed via western blot for pApaRs-His_(6x) (FIG. 3 b,bottom gel).

To perform western blots, clones, AOX2: A6 and B7; YPT1: D11 and B7;ICL1: E5 and H3; FLD1: E11 and F3; GAP: B7 and B10; and AOX1: E3 and E7were cultured under test expression conditions, pelletted (3000 g, 10min), and lysed with 2 ml YeastBuster (Novagen, Gibbstown, N.J.)+10 mMβ-mercaptoethanol and Complete Protease Inhibitor Cocktail tablets(Roche). Samples were cleared at 20,000 g and 15 μl of the lysate run ona 4-20% SDS-PAGE gel (1:15 h, 150 V). The protein was transferred to a0.45 micron nitrocellulose membrane (Bio-Rad) using a Trans-Blot SDsemi-dry transfer cell (Bio-Rad) in Tobin's transfer buffer (24 mM trisbase, 192 mM glycine, 20% ethanol) (2 h, 20 V, 100 mAmp). Residualprotein on gel was stained with Coomassie (FIG. 3 b, top) to ensureequal loading. The membrane was blotted using standard western blottingtechniques (Burnette (1981) “Western blotting: electrophoretic transferof proteins from sodium dodecyl sulfate-polyacrylamide gels tounmodified nitrocellulose and radiographic detection with antibody andradioiodinated Protein A.” Anal Biochem 112: 195-203) with an antiHis_(6x)-HRP conjugated antibody (Sigma-Aldrich) and detected with ECL(GE Healthcare) HRP chemiluminescence detection reagents and protocols(FIG. 3 b, bottom). Relative expression rates were determined by banddensity.

P_(FLD1) drove pApaRS transcription four-fold better than P_(AOX1) atthe mRNA level, and produced five-fold more pApaRS protein.P_(GAP),P_(YPT1),P_(ICL1), and P_(AOX2) all showed lower pApaRSexpression than P_(FLD1). Consistent with this result, the overall ambersuppression was highest with P_(FLD1)-pApa as measured by rHSA_(E37pApa)expression into the media (FIG. 4). As shown in FIG. 4, the two clonesfrom each promoter system were independently grown for six days withmethanol as the primary carbon source and pApa amino acid. 25 μl of thecleared media was run on a denaturing SDS-PAGE gel and stained withCoomassie. rHSA_(WT) (lane 15) was calculated to be 351.6 mg l⁻¹ by banddensity with BSA control. By density, P_(FLD1) (lanes 9 and 10 averaged)expressed 43% as much protein, or 151.2 mg l⁻¹. Maximum yields were >150mg L⁻¹ or approximately 43% of rHSA_(WT) yields (352 mg L⁻¹) (FIG. 11).FIG. 11 provides a bar graph representation of FIG. 4 showing ambersuppression in rHSA_(E37pApa) as a function of the promoter drivingpApaRS production. Protein production was determined by Coomassie banddensity on the SDS-PAGE gel shown in FIG. 4. rHSA_(WT) protein in FIG. 4was quantified as described as follows: 25 μl of BSA standards orunpurified rHSA_(WT) media from test protein expressions was run on anSDS-PAGE gel (see In FIG. 12). Lane 7 was a 1:1 dilution of therHSA_(WT) test protein expression media. BSA standard band densities(lanes 2-5) were plotted, and linearly fit. The densities for rHSA bands(2× lane 7 and lane 8) average to 83.33 or, 351.55 mg ml⁻¹. Yields ofunnatural protein (rHSA_(E37X) in other figures) were determined as apercentage of the same rHSA_(WT) sample.

The clones which produced most protein in FIG. 3 b were analyzed bynorthern blot for pApaRS mRNA transcription (FIG. 3 c bottom gel). Toperform the northern blots, top expressing clones AOX2, B7; YPT1, D11;ICL1, H3; FLD1, E11; GAP, B7; and AOX1, E3, were grown under testexpression conditions for 6 days. 3×10⁸ cells (2.5 ml at OD₆₀₀=1.0) werecollected and total RNA isolated via the RiboPure-Yeast Kit (Ambion)reagents and protocols. 13 μg of each RNA sample was loaded onto a 2%formaldehyde gel (2% agarose, 20 mM MOPS, 8 mM sodium acetate, 2.2 mMformaldehyde, pH=7.0). 3 volumes of NorthernMax formaldehyde load dye(Ambion) was mixed with 1 volume of RNA, heated to 65° C. for 15minutes, and chilled on ice for 5 minutes before loading. The gel waselectrophoresed (50 V for 2 h), and equal loading and RNA integrity wereconfirmed via ethidium bromide straining of 18S and 28S rRNA (FIG. 3 c,top). The RNA was drawn onto a Biodyne B nylon membrane (Pall LifeScience) in 10×SSC buffer (1.5 M sodium chloride, 0.15 M sodium citratepH=7.0) via a standard blotting apparatus. The membrane was rinsed in2×SSC buffer, dried, and auto-crosslinked with a UV Stratalinker 2400(Stratagene). Hybridization and detection was carried out via protocolsand reagents from the North2South Chemiluminescent Hybridization andDetection Kit (Pierce). Briefly, 400-500 μg of biotinylated probes:ketoRS3 biot 5′-/SBiosg/TGA GAC GCT GCT TAA CCG CTT C-3′ and ketoRS4biot 5′-/5Biosg/TAA AGA AGT ATT CAG GAT CGG ACT G-3′ were incubatedovernight at 55° C., bound to a streptavidin-HRP conjugate, and detectedwith a luminol/enhancer—stable peroxide solution (Pierce) (FIG. 3 c,bottom). Relative mRNA titers were determined by band density.

Oxime ligation to rHSA_(E37pApa)

To demonstrate the utility of this modified rHSA as a carrier forbioactive peptides, an oxime ligation was carried out between the uniqueketo side chain of rHSA_(E37pApa) and the anti-angiogenic peptideABT-510 (FIG. 5). A schematic representation of this ligation reactionis provided in FIG. 5 a. The ABT-510 peptide harbors anε-(2-(aminooxy)acetyl)-L-lysine as the sixth residue. Incubation of 75μM rHSA_(E37pApa) (FIG. 5 a, top reaction) with 2.25 mM peptideovernight at 37° C., as described in further detail below, results inthe formation of an oxime linkage. No reaction occurs with rHSA_(WT)(FIG. 5 a, bottom reaction) under identical conditions. Thisthrombospondin-1 (TSP-1) properdin type 1 repeat mimetic exhibits potentanti-tumor activity in humans, but suffers from rapid clearance by thekidneys when administered intravenously (Hoekstra et al. (2005) “Phase Isafety, pharmacokinetic, and pharmacodynamic study of thethrombospondin-1-mimetic angiogenesis inhibitor ABT-510 in patients withadvanced cancer.” J Clin Oncol 23: 5188-5197; Yang et al. (2007)“Thrombospondin-1 peptide ABT-510 combined with valproic acid is aneffective anti-angiogenesis strategy in neuroblastoma.” Cancer Res 67:1716-1724; Reiher et al. (2002) “Inhibition of tumor growth by systemictreatment with thrombospondin-1 peptide mimetics.” Int J Cancer 98:682-689). A nine amino acid peptide mimetic was synthesized with aunique ε-(2-(aminooxy)acetyl)-L-lysine UAA in place of the sixthL-norvaline residue (Anaspec, San Jose, Calif.). The sequence of thepeptide was: Ac-Sar-Gly-Val-D-aloIle-Thr-Lys(Aoa)-Ile-Arg-Pro-NEtMW=1097.3 Da). Based on the known structure-activity relationships ofTSP-1, modifications at this position are not expected to significantlyalter biological activity (Haviv et al. (2005) “Thrombospondin-1 mimeticpeptide inhibitors of angiogenesis and tumor growth: design, synthesis,and optimization of pharmacokinetics and biological activities.” J MedChem 48: 2838-2846). To perform the oxime ligation, 2.25 mM (0.5 mg) ofthe peptide was added to 75 μM rHSA_(E37pApa) or rHSA_(WT) (1.0 mg) in200 μl oxime ligation buffer (1.5 M sodium chloride, 500 mM sodiumacetate, pH=4.4) and incubated overnight at 37° C. At pH<5 the aminooxygroup undergoes a selective oxime ligation with the keto group of pApato covalently link the ABT-510 peptide to residue 37 of rHSA_(E37pApa)(FIG. 5 a, top reaction). The reactions were purified with a C8 VydacHPLC column (300 mm, 200 Å, 5 μm, Grace) on a Dynamax HPLC (Rainin)(elution 40-46% acetonitrile in water, 0.1%). Fractions were collected,combined, and analyzed via Coomassie stained SDS-PAGE gel. Intactprotein mass measurements were performed and the extent ofderivatization of rHSA_(E37pApa) with the peptide was confirmed using alinear MALDI-TOF MS Biflex III (Burker Daltonics, Billerica, Mass.)instrument with a sinapinic acid matrix to perform matrix assisted laserdesorption ionization (MALDI) mass spectrometry (FIG. 5 b). The mass ofrHSA_(WT) changed negligibly before and after treatment with theprotein, indicating that no conjugation was observed by MALDI massspectrometry when rHSA_(WT) (glutamic acid at residue 37) was treatedwith the amino-oxy modified ABT-510 peptide under identical conditions.

The mass difference between rHSA_(WT)+peptide andrHSA_(E37pApa)+peptide, less 60 Da owing to the E37pApa mutation (905 Daless 60 Da=845 Da), was used to determine ligation efficiency (˜77%).Previous conjugation protocols used an aniline catalyst for efficientligation (Dirksen et al. (2006) “Nucleophilic catalysis of oximeligation.” Angew Chem Int Ed Engl 45: 7581-7584; Dirksen et al. (2006)“Nucleophilic catalysis of hydrazone formation and transimination:implications for dynamic covalent chemistry.” J Am Chem Soc 128:15602-15603); however, oxime couplings to rHSA_(E37pApa) proceeded inapproximately 77% yield without the use of aniline in an overnightreaction using 75 μM rHSA_(E37pApa) and a thirty-fold excess of thepeptide.

Addition of 8 UAAs to the Genetic Repertoire

To illustrate the generality of this newly created recombinantexpression system, unnatural aaRSs evolved by the S. cerevisiaemethodology were inserted into pREAV-P_(FLD1). FIG. 6 a provides aschematic of the optimized pREAV-_(PFLD1) vector with E. coli tyrosyl-RSgene (aaRS) and tyrosyl suppressor tRNA cassette (tRNA_(CUA)). The aaRSsspecific for p-benzoylphenylalanine (pBpa, photocrosslinker, FIG. 6 b,Structure 3, Chin et al. (2003) “An expanded eukaryotic genetic code.“Science 301: 964-967); p-azidophenylalanine (pAzapa, photocrosslinker,chemically reactive, FIG. 6 b, Structure 4, Deiters et al. (2003)“Adding amino acids with novel reactivity to the genetic code ofSaccharomyces cerevisiae.” J Am Chem Soc 125: 11782-11783);p-(propargyloxy)phenylalanine (pPpa, chemically reactive, FIG. 6 b,Structure 5, Deiters et al. (2003) “Adding amino acids with novelreactivity to the genetic code of Saccharomyces cerevisiae.” J Am ChemSoc 125: 11782-11783); p-methoxyphenylalanine (pMpa, structure/functionprobe, FIG. 6 b, Structure 6, Chin et al. (2003) “An expanded eukaryoticgenetic code. “Science 301: 964-967); and p-iodophenylalanine (pIpa,heavy atom, FIG. 6 b, Structure 7, Chin et al. (2003) “An expandedeukaryotic genetic code.” Science 301: 964-967) were all inserted behindP_(FLD1) in the optimized pREAV-P_(FLD1) vector (FIGS. 6 a and 6 b). Forcomparison, wild type E. coli tyrosyl-RS (wt, FIG. 6 b, Structure 2) wasalso inserted into the new expression vector. Unnatural aaRSs specificfor tyrosine (wt), pBpa, pAzapa, pPpa, pMpa, and pIpa were amplified byPCR using the primers: KETO-Koz-F and KetoRS R 6×His (described above),digested with NcoI and EagI (NEB) and ligated into the similarlydigested pREAV-P_(FLD1)-pApaRS (after removal of the pApaRS region viaagarose gel purification). After sequence confirmation, the plasmidswere transformed into GS200-rHSA_(E37X) clone 1D12 as previouslydescribed to create GS200-rHSA_(E37X)/pREAV-P_(FLD1)-(synthetase_(tyr))(HIS4, ARG4, Gen^(R), Mut⁺). 12 clones were chosen from eachtransformation and screened via dot blot in 96 well format as previouslydescribed. The best producer was chosen from each (tyr, A9; pBpa, B7;pAzapa C9; pPpa, D6; pMpa, E6; and pIpa, F6) and compared to FLD1 (i.e.,the strain harboring pREAV-P_(FLD1)-pApaRS) E11 in rHSA expressionexperiments, as described above, to determine their abilities tosuppress the amber mutation at position 37 in rHSA_(E37X), where X isdefined as the unnatural amino acid) in the presence (+) and absence (−)of unnatural amino acids 1, 3-7 with their corresponding aaRS. Theresults of these experiments are depicted in FIG. 6 c. 25 μl ofunpurified cleared media was run on a SDS-PAGE gel and stained withCoomassie. Lane 2 is rHSA_(E37Y) expression with the wild type (wt)tyrosyl-RS. Lane 15 is expression of rHSA_(WT). Suppression yields weresimilar for the pApa and pAzapa mutants (40-45% the yield of rHSA_(WT));all other mutants with the exception of pIpa expressed >20% the yield ofrHSA_(WT) (FIG. 6 c). Relative protein yields were determined by banddensity. No protein expression was observed in the absence of thecognate amino acid, demonstrating the high orthogonality of this newsystem

Recently, a second orthogonal E. coli leucyl-derived RS/tRNA_(CUA) pair(aaRS denoted as LeuRS) was generated to incorporate additionalunnatural amino acids into proteins in S. cerevisiae (Lemke et al.(2007) “Control of protein phosphorylation with a genetically encodedphotocaged amino acid.” Nat Chem Biol 3: 769-772; Summerer et al. (2006)“A genetically encoded fluorescent amino acid.” Proc Natl Acad Sci USA103: 9785-9789). To accommodate unnatural LeuRSs derived from thisorthogonal pair in the new P. pastoris expression system, the tRNAregion of pREAV-P_(FLD1) plasmid was modified. The existing tRNA_(CUA)^(Tyr) cassette downstream of P_(PGK1) was excised and replaced by acoding region corresponding to three tandem repeats of tRNA_(CUA)^(Leu5) lacking the 5′ CCA and separated by SUP4 segments, as previouslydescribed, to create pREAV_(leu)-P_(FLD1). LeuRS mutants specific for4,5-dimethoxy-2-nitrobenzylserine (DMNB-S, photocaged serine, FIG. 6e,Structure 8, Lemke et al. (2007) “Control of protein phosphorylationwith a genetically encoded photocaged amino acid.” Nat Chem Biol 3:769-772) and2-amino-3-(5-(dimethylamino)naphthalene-Isulfonamide)propanoic acid(dansylalanine, dansyl fluorophore, FIG. 6e, Structure 9, Summerer etal. (2006) “A genetically encoded fluorescent amino acid.” Proc NatlAcad Sci USA 103: 9785-9789) were inserted behind P_(FLD1) to createpREAV_(leu)-P_(FLD1)-LeuRS (FIGS. 6 d and 6 f).

FIG. 6 d provides a schematic of the optimized pREAV_(leu)-P_(FLD1)vector with E. coli leucyl-RS gene and leucyl suppressor tRNA cassette(leu-tRNA (CUA). To create pREAV_(leu)-P_(FLD1) a section correspondingto three tandem repeats of tRNA_(CUA) ^(Leu5) lacking the 5′ CCA andseparated by SUP4 segments was synthesized (DNA 2.0, Menlo Park, Calif.)and PCR amplified using primers: Leu tRNA F, 5′-AAG GAA GCT AGC CTC TTTTTC AAT TGT ATA TGT G-3′ and Leu tRNA R, 5′-CGT ACA CGC GTC TGT ACA GAAAAA AAA GAA AAA TIT G-3′. The resulting 643 base pair product wasdigested with NheI and MluI (NEB) and ligated into the similarlydigested pREAV-P_(FLD1)-pApaRS (after removal of the tyrosyl tRNA viaagarose gel purification), to create pREAV_(leu)-P_(FLD1)-pApaRS. aaRSswith specificity for the DMNB-S and dansyl unnatural amino acids wereamplified using primers: LeuRS F, 5′-ATT CAC ACC ATG GAA GAG CAA TAC CGCCCG GAA GAG-3′ and LeuRS R, 5′-TTA ATT CGC GGC CGC TTA GCC AAC GAC CAGATT GAG GAG TTT ACC TG-3′, digested with NcoI and NotI (NEB), andligated into the similarly digested pREAV_(leu)-P_(FLD1)-pApaRS (afterremoval of the pApaRS coding region via agarose gel purification) tocreate pREAV_(leu)-P_(FLD1)-DMNB-S or pREAV_(leu)-P_(FLD1)-dansyl (FIG.6 d). After sequence confirmation, the plasmids were transformed intoGS200-rHSA_(E37X) (clone 1D12) and screened in 96 well dot blot formatas described. Clones A:A5 (DMNB-S) and B:G12 (dansyl) were identified assuccessful producers grown under test expression conditions for threedays post induction in buffer minimal methanol (BMM) media. rHSA_(WT)was expressed for three days in BMMY for comparison (FIG. 6 f). FIG. 6 fprovides the results of experiments performed to test the expression ofrHSA_(E37X) in the presence (+) and absence (−) of unnatural aminoacids. 25 μl of unpurified cleared media from each protein expressionwas analyzed on an SDS-PAGE gel and stained with Coomassie. Lane 4 isexpression of rHSA_(WT), also after three days

The LeuRS mutant specific for DMNB-S was recently shown to accept thecysteine analog of DMNB-S (DMNB-C), which was used in these expressionexperiments due to easier synthetic accessibility. Although smallamounts of full-length protein (a background of 35% of rHSA_(E37DMNB-S)for DMNB-S and 6% of rHSA_(E37dansyl) for dansyl) were produced in theabsence of the cognate amino acid, LC-MS/MS of a tryptic digestconfirmed high fidelity of the system in the presence of thecorresponding unnatural amino acid (FIG. 13). As shown in FIG. 13,rHSA_(E37DMNB-C) protein from lane 2 of FIG. 6 f was subjected totryptic digest followed by LC-MS/MS as described above, exceptchymotrypsin replaced trypsin in the digest. The top chromatogram(black) illustrates the total ion count (TIC) for the LC-MS/MS runbetween minutes 24.45, and 60.05. The third and fourth chromatograms areion extractions for the 2+ and 3+ charged species, respectively,corresponding to chymotryptic peptide, XDHVKLVNEVTEF, where X (the37^(th) residue of rHSA) is DMNB-C (total area under the peaks,“MA”=224582204). The fifth and sixth chromatograms are ion extractionsfor the 2+ and 3+ charged species, respectively, corresponding tochymotryptic peptide, XDHVKLVNEVTEF, where X is isoleucine of leucine(total area under the peaks “MA”=20029397). Calculations were done asfollows: percent E37DMNB-C=224582204/(224582204+20029397)*100=91.8% andpercent E37L=20029397/(224582204+20029397)*100=8.2%. Ion speciescorresponding to the incorporation of other natural amino acids at Xwere not detected in appreciable amounts.

Indeed, nonspecific readthrough of a nonsense codon is often suppressedby the presence of an aminoacylated suppressor tRNA. Suppression yieldswere approximately 37% the yield of rHSA_(WT) for rHSA_(E37DMNB-C) and23% the yield of rHSA_(WT) for rHSA_(E37dansyl) after three days ofexpression.

Previous attempts to optimize the expression of proteins containingunnatural amino acids in S. cerevisiae resulted in maximal yields of8-15 mg L⁻¹ in model systems, more than an order of magnitude less thandemonstrated in the P. pastoris system developed here. Work in the Wanglaboratory has recently shown that knockdown of the nonsense-mediatedmRNA decay (NMD) pathway in yeast can increase protein expression up to2-fold (Wang and Wang (2008) “New methods enabling efficientincorporation of unnatural amino acids in yeast.” J Am Chem Soc 130:6066-6067). Coupled with the use of a promoter derived from SNR52 todrive tRNA_(CUA) transcription, they were able to achieve yields300-fold higher yields of mutant protein than previously produced in S.cerevisiae, approximately 15 mg L⁻¹ (Wang and Wang (2008) “New methodsenabling efficient incorporation of unnatural amino acids in yeast.” JAm Chem Soc 130: 6066-6067). Thus, knockout of the UPF1 gene of the NMDpathway and use of the SNR52-tRNA_(CUA) promoter system may furtherincrease yields in P. pastoris. Additionally, work in the Kobayashilaboratory has demonstrated that yields of rHSA_(WT) from P. pastorisare more than an order of magnitude better (>10 g L⁻¹) when expressed infed-batch fermentation rather than in standard shake flasks (Ohya (2005)“Optimization of human serum albumin production in methylotrophic yeastPichia pastoris by repeated fed-batch fermentation.” Biotechnol Bioeng90: 876-887).

In summary, we have extended the methodology for the biosyntheticincorporation of unnatural amino acids into methylotrophic yeast. TwoaaRS/tRNA_(CUA) pairs were shown to be orthogonal in P. pastoris andused to express mutant proteins with

eight different unnatural amino acids in response to an amber codon atresidue 37 of rHSA_(E37X). Mutant proteins were expressed at high levelsin shake flasks with excellent fidelities. These results suggest thatthis expression system is amenable to many other unnatural amino acidswith synthetases currently being evolved in S. cerevisiae and is notlimited to the unnatural amino acids or aaRS/tRNA_(CUA) pairs discussedhere. The high yields and fidelities of this new system make it possibleto obtain useful amounts of therapeutic proteins with unique biologicaland pharmacological properties. For example, chemistries such as oximeligation or the copper catalyzed 1,3-cycloaddition reaction (“clickchemistry”) can be exploited to site-specifically PEGylate or crosslinkproteins, metal ion binding amino acids can be incorporated to bindradioisotopes, and peptide, toxin, or siRNA conjugates can be made fromcarrier proteins such as HSA or targeting proteins such as antibodies.In addition, the aforementioned rHSA_(E37pApa)-ABT-510 conjugates arebeing tested in in vitro anti-angiogenesis assays. The use ofrHSA_(E37pApa) as an endogenous, non-immunogenic carrier can be appliedto other rapidly cleared peptides including glucagon-like peptide 1mimetics (GLP-1) and parathyroid hormone (PTH) peptides.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

Amino acid sequence of mature HSA (not includingsignal sequence or propeptide sequence) SEQ ID NO: 1DAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF  E DHVKLVNEVTEFAKTCVAD ESAENCDKSLH TLFGDKLCT VATLRETYGEMADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTAFHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFTECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKFGERAFKAWAV ARLSQRFPKA EFAEVSKLVT DLTKVHTECCHGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEKSHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVFLGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADPHECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNALLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRMPCAEDYLSVV LNQLCVLHEK TPVSDRVTKC CTESLVNRRPCFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQTALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGLAmino acid sequence of mature TSP-1 (not including signal sequence)SEQ ID NO: 2 NR IPESGGDNSV FDIFELTGAA RKGSGRRLVK GPDPSSPAFRIEDANLIPPV PDDKFQDLVD AVRAEKGFLL LASLRQMKKTRGTLLALERK DHSGQVFSVV SNGKAGTLDL SLTVQGKQHVVSVEEALLAT GQWKSITLFV QEDRAQLYID CEKMENAELDVPIQSVFTRD LASIARLRIA KGGVNDNFQG VLQNVRFVFGTTPEDILRNK GCSSSTSVLL TLDNNVVNGS SPAIRTNYIGHKTKDLQAIC GISCDELSSM VLELRGLRTI VTTLQDSIRKVTEENKELAN ELRRPPLCYH NGVQYRNNEE WTVDSCTECHCQNSVTICKK VSCPIMPCSN ATVPDGECCP RCWPSDSADDGWSPWSEWTS CSTSCGNGIQ QRGRSCDSLN NRCEGSSVQTRTCHIQECDK RFKQDGGWSH WSPWSSCSVT CGDGVITRIRLCNSPSPQMN GKPCEGEARE TKACKKDACP INGGWGPWSPWDICSVTCGG GVQKRSRLCN NPTPQFGGKD CVGDVTENQICNKQDCPIDG CLSNPCFAGV KCTSYPDGSW KCGACPPGYSGNGIQCTDVD ECKEVPDACF NHNGEHRCEN TDPGYNCLPCPPRFTGSQPF GQGVEHATAN KQVCKPRNPC TDGTHDCNKNAKCNYLGHYS DPMYRCECKP GYAGNGIICG EDTDLDGWPNENLVCVANAT YHCKKDNCPN LPNSGQEDYD KDGIGDACDDDDDNDKIPDD RDNCPFHYNP AQYDYDRDDV GDRCDNCPYNHNPDQADTDN NGEGDACAAD IDGDGILNER DNCQYVYNVDQRDTDMDGVG DQCDNCPLEH NPDQLDSDSD RIGDTCDNNQDIDEDGHQNN LDNCPYVPNA NQADHDKDGK GDACDHDDDNDGIPDDKDNC RLVPNPDQKD SDGDGRGDAC KDDFDHDSVPDIDDICPENV DISETDFRRF QMIPLDPKGT SQNDPNWVVRHQGKELVQTV NCDPGLAVGY DEFNAVDFSG TFFINTERDDDYAGFVFGYQ SSSRFYVVMW KQVTQSYWDT NPTRAQGYSGLSVKVVNSTT GPGEHLRNAL WHTGNTPGQV RTLWHDPRHIGWKDFTAYRW RLSHRPKTGF IRVVMYEGKK IMADSGPIYDKTYAGGRLGL FVFSQEMVFF SDLKYECRDP Amino acid sequence of ABT-510 peptideSEQ ID NO: 3 (N-Ac-Sar)-Gly-Val-(D-alloIle)-Thr-Nva-Ile-Arg- (Pro-NHEt)

1. A method of making a covalently coupled carrier polypeptide-targetpolypeptide conjugate, the method comprising: incorporating a firstunnatural amino acid residue into a carrier polypeptide during synthesisor translation of the carrier polypeptide; incorporating a secondunnatural amino acid residue into a target polypeptide during synthesisor translation of the target polypeptide; and, reacting the first andsecond unnatural amino acid residues to produce the covalently coupledcarrier polypeptide-target polypeptide conjugate.
 2. The method of claim1, wherein incorporating the first unnatural amino acid into the carrierpolypeptide during translation comprises: (a) providing a translationsystem comprising (i) the first unnatural amino acid, (ii) an orthogonaltRNA-synthetase (O-RS), (iii) an orthogonal tRNA (O-tRNA) that isspecifically aminoacylated by the O-RS with the first unnatural aminoacid, and (iv) a nucleic acid encoding the carrier peptide, wherein thenucleic acid comprises a selector codon that is recognized by theO-tRNA; and, (b) translating the nucleic acid, thereby incorporating thefirst unnatural amino acid into the carrier polypeptide duringtranslation.
 3. The method of claim 1, wherein the carrier or targetpolypeptide is produced in a methylotrophic yeast cell.
 4. The method ofclaim 1, wherein the carrier or target polypeptide is produced in aCandida cell, a Hansenula cell, a Pichia cell, or a Torulopsis cell. 5.The method of claim 1, wherein the carrier polypeptide is homologous toa human serum albumin (HSA).
 6. The method of claim 1, wherein thecarrier polypeptide is or is homologous to an antibody, a HER2 antibody,and OKT3 antibody, an antibody fragment, an Fab, an Fc, an scFv, analbumin, a serum albumin, a bovine serum albumin, an ovalbumin, ac-reactive protein, a conalbumin, a lactalbumin, a keyhole limpethemocyanin (KLH), an ion carrier protein, an acyl carrier protein, asignal transducing adaptor protein, an androgen-binding protein, acalcium-binding protein, a calmodulin-binding protein, a ceruloplasmin,a cholesterol ester transfer protein, an f-box protein, a fattyacid-binding proteins, a follistatin, a follistatin-related protein, aGTP-binding protein, an insulin-like growth factor binding protein, aniron-binding protein, a latent TGF-beta binding protein, alight-harvesting protein complex, a lymphocyte antigen, a membranetransport protein, a neurophysin, a periplasmic binding protein, aphosphate-binding protein, a phosphatidylethanolamine binding protein, aphospholipid transfer protein, a retinol-binding protein, an RNA-bindingprotein, an s-phase kinase-associated protein, a sex hormone-bindingglobulin, a thyroxine-binding protein, a transcobalamin, a transcortin,a transferrin-binding protein, or a vitamin D-binding protein.
 7. Themethod of claim 1, wherein the target polypeptide is or is homologous toa TSP-1, an ABT-510, a glugacon-like peptide-1 (GLP-1), a parathyroidhormone (PTH), a ribosome inactivating protein (RIP), an angiostatin, anExedin-4, an apoprotein, an atrial natriuretic factor, an atrialnatriuretic polypeptide, an atrial peptide, a C-X-C chemokine, a T39765,a NAP-2, an ENA-78, a gro-a, a gro-b, a gro-c, an IP-10, a GCP-2, aNAP-4, a PF4, a MIG, a calcitonin, a c-kit ligand, a cytokine, a CCchemokine, a monocyte chemoattractant protein-1, a monocytechemoattractant protein-2, a monocyte chemoattractant protein-3, amonocyte inflammatory protein-1 alpha, a monocyte inflammatory protein-1beta, a RANTES, an I309, an R83915, an R91733, a T58847, a D31065, aT64262, a CD40 ligand, a complement inhibitor, a cytokine, an epithelialneutrophil activating peptide-78, a GROα, a MGSA, a GROβ, a GROγ, aMIP1-α, a MIP1-β, an MCP-1, an epithelial neutrophil activating peptide,an erythropoietin (EPO), an exfoliating toxin, a fibroblast growthfactor (FGF), an FGF21, a G-CSF, a gonadotropin, a growth factor, aHirudin, an LFA-1, a human insulin, a human insulin-like growth factor(hIGF), an hIGF-I, an hIGF-II, a human interferon, an IFN-α, an IFN-β,an IFN-γ, an interleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5,an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, akeratinocyte growth factor (KGF), a leukemia inhibitory factor, aneurturin, a PDGF, a peptide hormone, a pleiotropin, a pyrogenicexotoxin A, a pyrogenic exotoxin B, a pyrogenic exotoxin C, a relaxin, asomatostatin, a superoxide dismutase, a thymosin alpha 1, a human tumornecrosis factor (hTNF), a human tumor necrosis factor alpha, a humantumor necrosis factor beta, a Ras, a Tat, an inflammatory molecule, asignal transduction molecule, a bovine pancreatic trypsin inhibitor(BPTI), or a BP320 antigen.
 8. The method of claim 1, whereinincorporating the first unnatural amino acid into the carrierpolypeptide results in an HSA variant comprising the first unnaturalamino acid.
 9. The method of claim 8, wherein the first unnatural aminoacid is a p-acetylphenylalanine.
 10. The method of claim 9, wherein thefirst unnatural amino acid is incorporated into the HSA variant at aminoacid position 37, wherein numbering of amino acid position is relativeto that of SEQ ID NO:
 1. 11. The method of claim 9, wherein the firstunnatural amino acid is incorporated into the HSA variant duringtranslation.
 12. The method of claim 1, wherein incorporating the secondunnatural amino acid into the target polypeptide results in a TSP-1variant comprising the second unnatural amino acid.
 13. The method ofclaim 1, wherein incorporating the second unnatural amino acid into thetarget polypeptide results in an ABT-510 variant comprising the secondunnatural amino acid.
 14. The method of claim 12 or 13, wherein thesecond amino acid is an ε-(2-(aminooxy)acetyl)-L-lysine.
 15. The methodof claim 14, wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporatedinto the TSP-1 variant at amino acid position 6 wherein numbering ofamino acid position is relative to that of SEQ ID NO:
 2. 16. The methodof claim 14, wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporatedinto the TSP-1 variant at amino acid position 1 wherein numbering ofamino acid position is relative to that of SEQ ID NO:
 2. 17. The methodof claim 14, wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporatedinto the TSP-1 variant during synthesis.
 18. The method of claim 14,wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporated into theABT-510 variant at amino acid position 6 wherein numbering of amino acidposition is relative to that of SEQ ID NO:
 3. 19. The method of claim14, wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporated into theABT-510 variant at amino acid position 1 wherein numbering of amino acidposition is relative to that of SEQ ID NO:
 3. 20. The method of claim14, wherein the ε-(2-(aminooxy)acetyl)-L-lysine is incorporated into theABT-510 variant during synthesis.
 21. The method of claim 1, wherein thecarrier polypeptide is an HSA variant, wherein the first unnatural aminoacid is a p-acetylphenylalanine, wherein the p-acetylphenylalanine isincorporated into an HSA variant at amino acid position 37 duringtranslation, wherein the numbering of amino acid position in the HSAvariant is relative to SEQ ID NO: 1, wherein the target polypeptide is aTSP-1 variant, wherein the second unnatural amino acid isε-(2-(aminooxy)acetyl)-L-lysine, wherein theε-(2-(aminooxy)acetyl)-L-lysine is incorporated into the TSP-1 variantat amino acid position 6 during synthesis, wherein the numbering ofamino acid position in the TSP-1 variant is relative to SEQ ID NO: 2,and wherein the p-acetylphenylalanine and theε-(2-(aminooxy)acetyl)-L-lysine are reacted via oxime ligation toproduce an HSA-TSP-1 conjugate.
 22. The method of claim 1, wherein thecarrier polypeptide is an HSA variant, wherein the first unnatural aminoacid is a p-acetylphenylalanine, wherein the p-acetylphenylalanine isincorporated into an HSA variant at amino acid position 37 duringtranslation, wherein the numbering of amino acid position in the HSAvariant is relative to SEQ ID NO: 1, wherein the target polypeptide isan ABT-510 variant, wherein the second unnatural amino acid isε-(2-(aminooxy)acetyl)-L-lysine, wherein theε-(2-(aminooxy)acetyl)-L-lysine is incorporated into the ABT-510 variantat amino acid position 6 during synthesis, wherein the numbering ofamino acid position in the ABT-510 variant is relative to SEQ ID NO: 3,and wherein the p-acetylphenylalanine and theε-(2-(aminooxy)acetyl)-L-lysine are reacted via oxime ligation toproduce an HSA-ABT-510 conjugate.
 23. The method of claim 1, wherein thefirst and second unnatural amino acids are reacted via one or more of:an electrophile-nucleophile reaction, a ketone reaction with anucleophile, an oxime ligation, an aldehyde reaction with a nucleophile,a reaction between a carbonyl group and a nucleophile, a reactionbetween a sulfonyl group and a nucleophile, an esterification reaction,a reaction between a hindered ester group and a nucleophile, a reactionbetween a thioester group and a nucleophile, a reaction between a stableimine group and a nucleophile, a reaction between an epoxide group and anucleophile, a reaction between an aziridine group and a nucleophile, areaction between an electrophile and an aliphatic or aromatic amine, areaction between an electrophile and a hydrazide, a reaction between anelectrophile and a carbohydrazide, a reaction between an electrophileand a semicarbazide, a reaction between an electrophile and athiosemicarbazide, a reaction between an electrophile and acarbonylhydrazide, a reaction between an electrophile and athiocarbonylhydrazide, a reaction between an electrophile and asulfonylhydrazide, a reaction between an electrophile and a carbazide, areaction between an electrophile and a thiocarbazide, a reaction betweenan electrophile and a hydroxylamine, a reaction between a nucleophile ornucleophiles such as a hydroxyl or diol and a boronic acid or ester, atransition metal catalyzed reaction, a palladium catalyzed reaction, acopper catalyzed heteroatom alkylation reaction, a cycloadditionreaction, a 1,3, cycloaddition reaction, a 2,3 cycloaddition reaction,an alkyne-azide reaction, a Diels-Alder reaction, or a Suzuki couplingreaction.
 24. The method of claim 23, wherein said reaction is greaterthan 50% efficient, greater than 70% efficient, or greater than 90%efficient.
 25. A carrier polypeptide-target polypeptide conjugateproduced by the method of claim
 1. 26-40. (canceled)
 41. A carrierpolypeptide-target polypeptide conjugate comprising: a carrierpolypeptide domain comprising a first unnatural amino acid residue; and,a target polypeptide domain comprising a second unnatural amino acidresidue, wherein the carrier polypeptide domain and target polypeptidedomain are conjugated together through the first and second unnaturalamino acid residues. 42-46. (canceled)
 47. A cell comprising the carrierpolypeptide-target polypeptide conjugate of claim
 41. 48-52. (canceled)